U.S. patent application number 12/871669 was filed with the patent office on 2010-12-23 for compositions and methods for diagnosis and treatment of tumors.
This patent application is currently assigned to THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT. Invention is credited to PAUL K. GOLDSMITH, VIVIAN TAKAFUJI, EDWARD J. UNSWORTH, XIN WEI WANG.
Application Number | 20100322950 12/871669 |
Document ID | / |
Family ID | 38834371 |
Filed Date | 2010-12-23 |
United States Patent
Application |
20100322950 |
Kind Code |
A1 |
TAKAFUJI; VIVIAN ; et
al. |
December 23, 2010 |
COMPOSITIONS AND METHODS FOR DIAGNOSIS AND TREATMENT OF TUMORS
Abstract
Based on the observation of the cooperation of osteopontin (OPN)
and matrixmetalloproteinase-9 (MMP-9) in the promotion of the
metastatic phenotype, therapies and diagnostic assays are disclosed
for the treatment of a tumor that overexpresses OPN, such as
hepatocellular carcinoma (HCC), for example metastatic HCC. In one
example, methods of treating a tumor include administration of an
agent that reduces cellular invasion resulting from the interaction
between a fragment of OPN (OPN-5 kD) generated by MMP-9 cleavage
and CD44 receptor. Examples of such agents include fragments of
OPN-5 kD and antibodies specific for OPN-5 kD. Therapeutic
compositions are also provided that include such agents. Also
provided are methods of diagnosing or prognosing a tumor, for
example by detecting expression of OPN-5 kD peptide or OPN-c mRNA
in a biological sample obtained from the subject. Also provided are
antibodies that specifically bind OPN-5 kD.
Inventors: |
TAKAFUJI; VIVIAN;
(ALEXANDRIA, VA) ; WANG; XIN WEI; (ROCKVILLE,
MD) ; UNSWORTH; EDWARD J.; (WESTPORT, MA) ;
GOLDSMITH; PAUL K.; (ROCKVILLE, MD) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP (OTT-NIH)
121 S.W. SALMON STREET, SUITE #1600
PORTLAND
OR
97204-2988
US
|
Assignee: |
THE GOVERNMENT OF THE UNITED STATES
OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE
DEPARTMENT
OF HEALTH AND HUMAN SERVICES
|
Family ID: |
38834371 |
Appl. No.: |
12/871669 |
Filed: |
August 30, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12340211 |
Dec 19, 2008 |
7803380 |
|
|
12871669 |
|
|
|
|
PCT/US2007/071712 |
Jun 20, 2007 |
|
|
|
12340211 |
|
|
|
|
60805298 |
Jun 20, 2006 |
|
|
|
Current U.S.
Class: |
424/174.1 ;
435/252.3; 435/254.2; 435/320.1; 435/344.1; 435/375; 435/419;
435/6.14; 435/7.92; 436/501; 530/387.3; 530/387.9; 530/388.85;
530/391.7; 536/23.53 |
Current CPC
Class: |
C07K 16/2884 20130101;
G01N 33/57484 20130101; A61P 35/00 20180101; C07K 16/18 20130101;
C07K 16/40 20130101; C07K 2317/76 20130101; C07K 16/24 20130101;
G01N 33/57438 20130101; A61K 2039/505 20130101; A61K 38/00
20130101; C07K 16/2848 20130101; G01N 2333/52 20130101 |
Class at
Publication: |
424/174.1 ;
436/501; 435/6; 435/375; 435/344.1; 530/388.85; 530/387.9;
530/387.3; 530/391.7; 536/23.53; 435/320.1; 435/252.3; 435/419;
435/254.2; 435/7.92 |
International
Class: |
A61K 39/395 20060101
A61K039/395; G01N 33/53 20060101 G01N033/53; C12Q 1/68 20060101
C12Q001/68; C12N 5/00 20060101 C12N005/00; C12N 5/16 20060101
C12N005/16; C07K 16/18 20060101 C07K016/18; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 1/21 20060101
C12N001/21; C12N 5/10 20060101 C12N005/10; C12N 1/19 20060101
C12N001/19; A61P 35/00 20060101 A61P035/00; G01N 33/00 20060101
G01N033/00 |
Claims
1. A method of diagnosing an osteopontin (OPN)-overexpressing tumor
in a subject, comprising: detecting an OPN-5 kD fragment in a
sample obtained from the subject, wherein detection of the OPN-5 kD
fragment in the sample indicates that the subject has an
OPN-expressing tumor.
2. The method of claim 1, wherein the OPN-5 kD fragment consists of
a peptide sequence of 40 to 50 amino acids comprising at least 95%
sequence identity to the sequence shown in SEQ ID NO: 4.
3. The method of claim 1, further comprising comparing the detected
OPN-5 kD fragment to a control, wherein the control is a value of
OPN-5 kD fragment expected if the subject does not have an
OPN-expressing tumor, and wherein an at least 1.5-fold increase in
the detected OPN-5 kD fragment in the sample relative to the
control indicates that the subject has an OPN-expressing tumor.
4. The method of claim 1, wherein detecting the OPN-5 kD fragment
in a sample comprises: contacting the sample with an OPN-5 kD
antibody under conditions wherein an immune complex will form; and
detecting the formation of the immune complex, wherein the presence
of the immune complex detects the presence of the OPN-5 kD
fragment.
5. The method of claim 1, wherein the sample comprises blood or a
fraction thereof.
6. The method of claim 1, wherein the OPN-expressing tumor
comprises hepatocellular carcinoma (HCC).
7. The method of claim 6, wherein the HCC is a metastasis.
8. The method of claim 7, wherein the metastasis is an
intra-hepatic metastasis.
9. The method of claim 7, wherein the metastasis is an
extra-hepatic metastasis.
10. A method of diagnosing an OPN-expressing tumor in a subject,
comprising: determining an amount of OPN-c expression in a tumor
sample obtained from the subject; determining an amount of OPN-a
expression in a tumor sample obtained from the subject; and
comparing the amount of OPN-c expression in the tumor sample to the
amount of OPN-a expression in the tumor sample, wherein a
statistically significant increase in OPN-c expression in the tumor
sample compared to the amount of OPN-a expression in the tumor
sample indicates that the subject has an OPN-expressing tumor.
11. The method of claim 10, further comprising determining whether
the tumor expresses a greater level of OPN-c mRNA than adjacent
non-tumor tissue, wherein a statistically significant increase in
OPN-c mRNA expression in the tumor sample compared to the adjacent
non-tumor tissue indicates that the subject has a tumor with
increased metastatic potential.
12. A method of determining the metastatic potential of an
OPN-expressing tumor in a subject, comprising: determining an
amount of OPN-c expression in a tumor sample obtained from the
subject; determining an amount of OPN-c expression in a sample
obtained adjacent to the tumor from the subject; and comparing the
amount of OPN-c expression in the tumor sample to the adjacent
sample, wherein a statistically significant increase in OPN-c
expression in the tumor sample compared to the adjacent sample
indicates that the subject has an OPN-expressing tumor with
increased metastatic potential.
13. A method for inhibiting the growth an OPN-expressing tumor
cell, comprising contacting the cell with an OPN-5 kD specific
antibody, thereby inhibiting the growth of the OPN-expressing tumor
cell.
14. The method of claim 13, wherein the OPN-expressing tumor cell
is in a subject that has an OPN-expressing tumor and the method is
a method of treating the OPN-expressing tumor, and contacting the
cell comprises administering to the subject a therapeutically
effective amount of the OPN-5 kD specific antibody, thereby
treating the OPN-expressing tumor.
15. The method of claim 14, wherein serum from the subject has
detectable OPN-5 kD fragment, wherein the tumor expresses a greater
amount of OPN-c mRNA than OPN-a mRNA, or both.
16. The method of claim 14, wherein the method further comprises
administering one or more additional therapeutic agents at a
therapeutically effective amount to the subject.
17. The method of claim 14, further comprising determining whether
the subject has significantly increased levels of an OPN-5 kD
fragment in its serum compared to a subject not having a tumor,
wherein the presence of significantly increased levels of the OPN-5
kD fragment in the serum indicates that the subject has an
OPN-expressing tumor.
18. An isolated hybridoma cell line 5 kd106-13 D that produces a
monoclonal antibody that specifically binds an OPN-5 kD
fragment.
19. An isolated monoclonal antibody produced by the hybridoma cell
line of claim 18, a chimeric form thereof, or a humanized form
thereof, or a functional fragment thereof, wherein isolated
monoclonal antibody, the chimeric form thereof, humanized form
thereof, or the functional fragment thereof specifically binds the
OPN-5 kD fragment.
20. An isolated antibody that specifically binds to an OPN-5 kD
fragment epitope sequence EELNGAY (amino acids 30 to 36 of SEQ ID
NO: 4).
21. The isolated antibody of claim 20, wherein the antibody is a
monoclonal antibody, a chimeric form thereof, a humanized form
thereof, or a functional fragment thereof, wherein isolated
monoclonal antibody, chimeric form thereof, humanized form thereof,
or a functional fragment thereof specifically binds the OPN-5 kD
fragment.
22. An isolated chimeric form of the monoclonal antibody of claim
21 or functional fragment thereof, wherein the isolated chimeric
form or a functional fragment thereof comprises the CDRs of the
isolated monoclonal antibody of claim 21.
23. An isolated humanized form of the monoclonal antibody of claim
21 or functional fragment thereof, wherein the isolated humanized
form or a functional fragment thereof comprises the CDRs of the
isolated monoclonal antibody of claim 21.
24. An isolated functional fragment of the monoclonal antibody of
claim 21.
25. The isolated monoclonal antibody, chimeric form thereof,
humanized form thereof, or functional fragment thereof of claim 21,
conjugated to an effector molecule.
26. An isolated nucleic acid encoding the monoclonal antibody,
chimeric form thereof, humanized form thereof, or functional
fragment thereof of claim 21.
27. The isolated nucleic acid of claim 26, operably linked to a
promoter.
28. An isolated expression vector comprising the nucleic acid of
claim 27.
29. An isolated host cell transformed with the expression vector of
claim 28.
30. A composition comprising an effective amount of the monoclonal
antibody, chimeric form thereof, humanized form thereof, or
functional fragment thereof of claim 21.
31. A kit, comprising: the isolated antibody of claim 20; and one
or more agents that permit detection of binding of the antibody to
OPN-5 kD in a biological sample and/or at least one anti-neoplastic
agent.
32. A method of diagnosing an OPN-expressing tumor in a subject,
comprising contacting a sample from the subject with the isolated
antibody of claim 20; and detecting binding of the antibody to the
sample, wherein an increase in the binding of the antibody to the
sample as compared to a control indicates that the subject has an
OPN-expressing tumor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. application Ser. No. 12/340,211
filed on Dec. 19, 2008 now U.S. Pat. No. ______, which is a
continuation-in-part of International Patent Application No.
PCT/US2007/071712 filed on Jun. 20, 2007, which claims priority
from U.S. Provisional Application No. 60/805,298 filed Jun. 20,
2006, which is herein incorporated by reference.
FIELD
[0002] This application relates to methods of diagnosing and
treating a tumor that has increased expression of osteopontin
(OPN), such as metastatic hepatocellular carcinoma (HCC), as well
as compositions and kits that can be used for such methods. Also
provided are antibodies that specifically bind to a fragment of
OPN(OPN-5 kD) generated by metalloproteinase-9 (MMP-9)
cleavage.
BACKGROUND
[0003] Elucidating the molecular events that promote tumor cell
invasion continues to be a challenge for the treatment and
prevention of hepatocellular carcinoma (HCC).
[0004] HCC is a highly aggressive carcinoma of the liver which has
been reported to occur world-wide in increasing numbers.
Intra-hepatic metastatic recurrences via the portal vein are the
main cause of death in HCC patients who have undergone partial
hepatectomy or liver transplantation (Korn, World J.
Gastroenterol., 7:777-8, 2001). Identifying the main `players` and
how they contribute to the tumor cell metastatic cascade, for
example at the early stages of cellular invasion, can present
opportunities for lessening the severity of HCC through new
therapeutic interventions.
[0005] Osteopontin (OPN, SPP1) is a secreted multi-functional
glycoprotein expressed at high levels in tumors and the surrounding
stroma of numerous cancers, including those of the liver, and is
alternatively spliced into at least 3 isoforms (OPN-a, OPN-b,
OPN-c) (Butler, Ann. N.Y. Acad. Sci., 760:6-11, 1995; Coppola et
al., Clin. Cancer Res., 10:184-90, 2004). Increased serum and
plasma OPN levels (.about.4-10 fold) are associated with advanced
stage lung, hepatic, breast, colon, and prostate carcinomas (Oates
et al., Invasion Metastasis, 17:1-15, 1997; Fedarko et al., Clin.
Cancer Res., 7:4060-6, 2001; Singhal et al., Clin. Cancer Res.,
3:605-11, 1997). OPN expression can predict high grade, late stage
and early recurrence HCC (Pan et al., Cancer, 98:119-27, 2003) and
is highly correlated with tumor recurrence and decreased patient
survival following orthotopic liver transplantation (Wang,
Hepatology, 42(4), Suppl. 1:391 A, 2005). Prominent OPN levels have
been detected in metastatic HCC tumor cells at the leading edge of
pseudopodia and filopodia (Suzuki et al., J. Bone Miner. Res.,
17:1486-97, 2002) and in macrophages at the tumor-stroma interface
(Senger et al., Ann. N.Y. Acad. Sci., 760: 83-100, 1995). A
correlation between OPN mRNA expression and primary HCC tumor
metastasis has been shown (Ye et al., Nat. Med., 9: 416-423, 2003).
Cytoplasmic OPN was detected in vascularized regions of primary HCC
tumors but not in normal liver. In addition, a neutralizing
antibody to OPN decreased pulmonary secondary lesions in nude mice
and inhibited tumor cell invasion.
[0006] Local and extra-hepatic HCC tumor cell invasion is
associated with extensive matrix remodeling, angiogenesis, and
hepatocyte injury (McKenna et al., Am. J. Surg., 183: 588-94,
2002). The members of the zinc-dependent endopeptidase family of
matrix metalloproteinases (MMPs) catabolize extracellular matrix
components (Theret et al., Hepatology, 34:82-88, 2001; Liaw and
Crawford, Braz. J. Med. Biol. Res., 32:805-812, 1999). Each MMP
contains a catalytic and pro-peptide regulatory domain and a
variable number of carboxy-terminal hemopoexin-like structural
domains and are broadly divided into subclasses based on substrate
activity. The two gelatinases (MMP-2 and MMP-9) play roles in tumor
invasion and angiogenesis and participate in cancer progression in
several neoplasias (Turpeenniemi-Hujanen, Biochimie, 87:287-97,
2005; Hanemaaijer et al., Int. J. Cancer, 86:204-7, 2000; Scorilas
et al., Br. J. Cancer, 84:1488-96, 2001). Active MMP-9
enzymatically cleaves proteins of the basement membrane (such as
collagens type IV, V, VII, X, and XIV) and can be detected at the
invasive front of HCC (Kaneyoshi et al., Clin. Cancer Res.,
7:4027-32, 2001). A substantial increase in MMP-9 mRNA levels in
HCC primary metastatic tumors has been observed (Ye et al., Nat.
Med. 9:416-23, 2003), which is consistent with indications of HCC
tumor malignancy and MMP-9 abundance (Ashida et al., Am. J.
Pathol., 149:1803-11, 1996; Wei et al., Hunan. Yi. Ke. Da. Xue.
Xue. Bao., 28:212-6, 2003).
[0007] Stromelysin-1 (MMP-3) and matrilysin (MMP-7) are reported to
cleave OPN at residues 166 and 210 (Agnihotri et al., J. Biol.
Chem., 276:28261-7, 2001). MMP-3/-7 digested OPN fragments increase
AsPC-1 and HeLa tumor cell adhesion via cell surface integrin
receptors and MMP-3 cleaved OPN can increase mouse peritoneal
macrophage cell migration. The thrombin coagulation factor also
cleaves OPN at residue 168, resulting in two fragments of similar
molecular weight (.about.28-30 kD) can be detected in the serum and
plasma of patients with cancer (Senger et al., Cancer Res.,
48:5770-4, 1988). Thrombin-cleaved OPN can mediate increased tumor
and macrophage cell adhesion and migration via exposure of the
amino-terminal reactive RGD sequence and binding to cell surface
integrins, namely the vitronectin receptor .alpha.V.beta.3,
although interactions with .alpha.V.beta.1, .alpha.V.beta.5,
.alpha.4.beta.1, and .alpha.9.beta.1 have been described (Sodek et
al., Crit. Rev. Oral Biol. Med., 11:279-303, 2000; Wai and Kuo, J.
Surg. Res., 121:228-41, 2004; Weber, Biochim. Biophys. Acta,
1552:61-85, 2001). Conversely, the COOH-terminal thrombin-cleaved
fragment has been proposed to induce macrophage migration primarily
through CD44 receptors (Weber et al., J. Leukoc. Biol., 72:752-61,
2002).
SUMMARY
[0008] Methods of treating a tumor that overexpresses osteopontin
(OPN), such as a tumor that overexpresses OPN (for example OPN-c)
and MMP-9, as well as diagnosing and prognosing such tumors, are
provided. Examples of such tumors include but are not limited to
cancers of the liver, breast, colon, and prostate. The disclosed
treatment, diagnostic, and prognostic methods can be used in
combination or individually.
[0009] The inventors have determined that MMP-9 mediates OPN
proteolytic cleavage into several fragments including OPN-5 kD
(such as SEQ ID NO: 4), which is a ligand for CD44 receptor. The
interaction of OPN-5 kD and the CD44 receptor increases HCC tumor
cell invasion. The inventors have identified several fragments of
OPN-5 kD that significantly reduce HCC tumor cell invasion, which
may be achieved by disrupting the interaction between OPN-5 kD and
CD44 receptor. Based on this observation, new methods of treating
such tumors are disclosed, for example by using short
invasive-blocking peptides targeted to the effects of OPN-5 kD. The
inventors have also identified monoclonal antibodies that bind to
OPN-5 kD which can be used to significantly reduce HCC tumor cell
invasion by disrupting the interaction between OPN-5 kD and the
CD44 receptor. Therefore, new methods of treating such tumors are
disclosed, for example by administering therapeutically effective
amounts of one or more peptides or antibodies that reduce or
inhibit the effects of OPN-5 kD.
[0010] Methods of treating a tumor in a subject are provided, such
as a tumor that overexpresses OPN, MMP-9, or both. A particular
example of such a tumor is HCC. In one example, the method includes
administering to the subject a therapeutically effective amount of
an agent that decreases cellular invasion of a tumor cell effected
by the interaction of OPN-5 kD and the CD44 receptor, such as a
peptide fragment of OPN-5 kD, or an antibody that binds to OPN-5
kD, thereby treating the tumor. A particular example of OPN-5 kD
sequence is provided in SEQ ID NO: 4. However, one skilled in the
art will appreciate that variants of this sequence (such as
polymorphisms) can retain OPN-5 kD activity (such as the ability to
bind to CD44 receptor with high affinity). For example, a variant
OPN-5 kD sequence may include at least one amino acid deletion,
substitution, addition, or combinations thereof, such as 1-5
conservative amino acid substitutions, while retaining the ability
to stimulate cellular invasion upon interaction with the CD44
receptor.
[0011] Exemplary therapeutic peptides can include at least one
peptide sequence, or a plurality of peptide sequences, wherein each
peptide fragment of OPN-5 kD includes at least 5 contiguous amino
acids of OPN-5 kD and in some examples is no more than 50 amino
acids. For example, the peptide can be a composition that includes
one or more peptides consisting of the amino acid sequence shown in
SEQ ID NO: 5, 6, 7, or 8. Exemplary therapeutic antibodies can
include a single monoclonal antibody or an equivalent specific
binding agent the binds to one epitope (e.g., amino acids 30-36 of
SEQ ID NO: 4) or multiple antibodies that bind to more than one
epitope in OPN-5 kD. If desired, therapeutically effective amounts
of one or more additional therapeutic agents, such as an
anti-neoplastic chemotherapeutic agent, can be administered to the
subject. In particular examples, treatment includes reducing
cellular invasion by a tumor, such as reducing or preventing
metastasis of a tumor. In some examples, treating the tumor
prolongs survival time of the subject (for example by at least 2
months, at least 6 months, or even at least 12 months).
[0012] Also provided by the present disclosure are therapeutic
compositions that can be used to treat a tumor, such as a tumor
that overexpresses OPN (such as overexpresses OPN and MMP-9), for
example HCC. Such compositions can be used in the therapeutic
methods provided herein. In one example, the composition includes
one or more peptide fragments of OPN-5 kD. In a specific example,
the composition includes one or more peptides consisting of the
amino acid sequence of SEQ ID NO: 5, 6, 7, or 8. In another example
the therapeutic compositions include antibodies that specifically
bind to OPN-5 kD. The disclosed compositions can include one or
more pharmaceutically acceptable carriers, as well as additional
therapeutic agents (such as other anti-neoplastic agents). Such
therapeutic compositions can also be part of a kit, such as a kit
that includes one more other anti-neoplastic agents (such as IL-2,
IL-12, GM-CSF, a chemotherapeutic agent, or combinations
thereof).
[0013] Methods of diagnosing and prognosing a tumor (such as a
tumor that overexpresses OPN, MMP-9, or both) are provided. In some
examples, such methods are performed prior to the treatment methods
described herein. However, such methods can also be used
independently of the disclosed treatment methods. In particular
examples, the method includes determining if the OPN-5 kD fragment
is present in the subject, for example by detecting OPN-5 kD in the
subject's serum. For example, a sample from the subject that
includes peptides (such as serum) is contacted with an agent that
specifically binds to OPN-5 kD (such as an antibody that
specifically binds to an epitope of OPN-5 kD), then it is
determined whether the agent specifically bound to proteins in the
sample. If specific binding of the agent to OPN-5 kD in the sample
is detected, this indicates that the subject has a tumor (such as
HCC), has a poor prognosis (for example because this indicates that
the tumor has metastasized), or both. Such a method provides a
non-invasive means for diagnosis and prognosis of a tumor, such as
metastatic HCC. In another example, the method includes contacting
a sample from the subject that includes nucleic acid molecules
(such as a tumor sample) with an agent that permits detection of
OPN-c nucleic acid molecules (such as cDNA or mRNA), then
determining a relative amount of OPN-c nucleic acid molecules in
the sample. For example, if increased OPN-c mRNA expression is
detected in non-cancerous tissue adjacent to the tumor or in the
tumor itself (or both), this indicates that the subject's tumor has
increased metastatic potential. Similar methods can be used to
measure OPN-c protein expression, as an alternative to OPN-c
nucleic acid molecule expression.
[0014] Also provided are antibodies that specifically bind OPN-5
kD, such as an antibody that binds to an epitope sequence within
SEQ ID NO: 4, or a fragment thereof such as any of SEQ ID NOS: 5-8.
A specific epitope is amino acids 30-36 of SEQ ID NO: 4. Such
antibodies are useful for detection and treatment of tumors and can
be part of a kit. Diagnostic kits can include other agents to
permit detection of the antibody, such as a labeled secondary
antibody. A specific example of such antibodies is the OPN-5 kD
monoclonal antibody 5 kd106-13 D. Thus, the 5 kd106-13 D antibody,
chimeric form or humanized form thereof or functional fragment
thereof can be used to detect OPN-5 kD and OPN-expressing tumors,
as well as treat such tumors. Chimeric forms of 5 kd106-13 D,
humanized forms of 5 kd106-13 D, and functional fragments of 5
kd106-13 D, are also disclosed. The 5 kd106-13 D antibody, chimeric
form, humanized form or functional fragment of these antibodies can
be conjugated to an effector molecule, such as a detectable marker,
a therapeutic agent, or a toxin. Kits that contain the monoclonal
antibody 5 kd106-13 D, a chimeric form, or a humanized form thereof
or functional fragments thereof are also disclosed. Nucleic acid
molecules encoding the monoclonal antibody 5 kd106-13 D, a chimeric
form or humanized form thereof or functional fragments thereof are
also disclosed. A hybridoma that produces 5 kd106-13 D is also
provided.
[0015] The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A are two plots showing that mRNA expression of OPN
(left) and MMP-9 (right) correlate with HCC tumor metastasis.
NM=non-metastatic (n=11), M=metastatic (n=14), MM=metastatic
intra-hepatic lesion (n=10). All data were normalized to mRNA
levels detected in the normal tissue immediately adjacent to the
tumor samples (T/N). Statistical analysis was performed using Mann
Whitney non-parametric t-tests (.alpha.=0.05).
[0017] FIG. 1B is a graph showing OPN and MMP-9 expression level
correlation as determined using linear regression analysis after
plotting values within each patient case.
[0018] FIG. 2A is a digital image of a Western blot showing a
time-dependent MMP-9-cleavage of recombinant human OPN (.about.65
kD) (lanes 2-6). MMP inhibitors were added to separate reactions
and analyzed after a 1-hour digestion at 37.degree. C.: EDTA (10
mM), MMP-9 inhibitor I (2 .mu.M) and TIMP-1 (0.1 .mu.M) (lanes 8,
10, 11 respectively). Undigested OPN (lane 1), OPN exposed to
thrombin cleavage (0.05 U) (lane 7) and TIMP-1 alone lanes (9) were
included as controls.
[0019] FIG. 2B is a graph showing the kinetics of the MMP-9
specific OPN cleavage quantified by densitometric analysis of the
three bands marked by arrows in FIG. 2A and presented as a fraction
of starting values at Time=0.
[0020] FIG. 3A is a schematic drawing showing the expression
vectors generated to determine the activity level of OPN
fragments.
[0021] FIG. 3B is a digital image of a Western blot showing
expression of the four constructs in FIG. 3A in HEK 293 cells.
[0022] FIG. 4A is a digital image of a Western blot (left) and a
bar graph showing densitometric analysis of OPN-5 kD expression in
FIG. 4A (right), demonstrating that endogenous OPN protein levels
correlate with HCC cell line metastatic potential. Data were
adjusted relative to Hep3B levels (=1) following normalization to
beta-actin levels.
[0023] FIG. 4B is a digital image of a protein gel showing
gelatinase activity relative to pro-MMP-9, active MMP-9, and active
MMP-2 standards.
[0024] FIG. 4C is a bar graph showing the percent cell invasion for
three HCC cell lines. Data are presented as the mean percent cell
invasion and standard deviation following normalization to
fluorescence readings corresponding to cells migrating through
uncoated control membranes.
[0025] FIG. 5A is a bar graph showing that MMP-9-cleaved OPN
increased HCC cell adhesion relative to uncleaved OPN. Student
un-paired t-tests were used to compare cleaved versus un-cleaved
OPN adhesion (.alpha.=0.05).
[0026] FIG. 5B is a bar graph showing adhesion of HCC cells in
response to four OPN constructs. Student un-paired t-tests were
used to compare media versus OPN adhesion at each time point
(.alpha.=0.05).
[0027] FIG. 5C is a graph showing the effect of different length
OPN molecules on migration of SMMC-7721. Data are presented
relative to vector-transfected cell controls at each of the time
points. * denotes significance p<0.017.
[0028] FIG. 6A is a bar graph showing that the OPN-5 kD fragment
induced reproducible increases in cellular invasion. Data are
presented as the mean percent cell invasion and standard deviation
following normalization to fluorescence readings corresponding to
cells migrating through uncoated control membranes. Un-paired
student's t-tests were used to compare mean and standard deviation
values (.alpha.=0.05).
[0029] FIG. 6B is a bar graph showing the effect of the presence of
blocking antibodies to the integrin .alpha.V.beta.3 and CD44
receptors on the invasion of HCC cells transiently expressing OPN
full-length or OPN-5 kD. Un-paired student's t-tests were used to
compare mean and standard deviation values (.alpha.=0.05).
[0030] FIG. 6C is a bar graph showing the effect of varying
concentrations of the OPN-5 kD peptide on the invasion of HCC
cells. Un-paired student's t-tests were used to compare mean and
standard deviation values of media versus treated cells
(.alpha.=0.05).
[0031] FIGS. 7A and B are bar graphs showing that some 10-mer
peptides can inhibit OPN-5 kD induced HCC cellular invasion. Data
were normalized to uncoated membrane chamber values and adjusted by
media control values (=1). (A) Un-paired student's t-tests were
used to compare mean and standard deviation values of OPN-5 kD
peptide versus each of the 10-mer peptides (.alpha.=0.05). (B)
Significance of the small peptide inhibition of the OPN-5 kD
peptide increased cellular invasion was tested using un-paired
student's t-tests (.alpha.=0.05). * denotes significance (A)
p<0.037 and (B) p<0.002.
[0032] FIGS. 8A and 8B are plots showing the OPN splice variants
expressed in (A) tumor or (B) non-cancerous tissue. Data are shown
following normalization to 18S rRNA (Applied Biosystems, CA) and
relative to a normal liver tissue reference pool (n=8).
[0033] FIG. 9 is a digital image of a western blot showing
detection of OPN-5 kD with the monoclonal antibody 5 kd106-13
D.
SEQUENCE LISTING
[0034] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and one letter code for amino
acids. Only one strand of each nucleic acid sequence is shown, but
the complementary strand is understood as included by any reference
to the displayed strand.
[0035] SEQ ID NO: 1 is a cDNA sequence for a human
osteopontin-a.
[0036] SEQ ID NO: 2 is the protein encoded by SEQ ID NO: 1.
[0037] SEQ ID NO: 3 is a cDNA sequence for a human
osteopontin-c.
[0038] SEQ ID NO: 4 is an amino acid sequence of OPN-5 kD.
[0039] SEQ ID NO: 5 is the p3 fragment of OPN-5 kD.
[0040] SEQ ID NO: 6 is the p6 fragment of OPN-5 kD.
[0041] SEQ ID NO: 7 is the p7 fragment of OPN-5 kD.
[0042] SEQ ID NO: 8 is a fragment of OPN-5 kD.
[0043] SEQ ID NO: 9 is the protein encoded by SEQ ID NO: 3.
[0044] SEQ ID NO: 10 is a unique reporter sequence for OPN-a.
[0045] SEQ ID NO: 11 is a unique reporter sequence for OPN-c.
[0046] SEQ ID NOS: 12-15 show exemplary light chain frameworks of
human MAb LEN.
[0047] SEQ ID NOS: 16-19 show exemplary heavy chain frameworks of
human MAb 21/28 CL.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Abbreviations and Terms
[0048] The following explanations of terms and methods are provided
to better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. The singular forms "a," "an," and "the" refer to one or
more than one, unless the context clearly dictates otherwise. For
example, the term "comprising a peptide" includes single or plural
peptides and is considered equivalent to the phrase "comprising at
least one peptide." The term "or" refers to a single element of
stated alternative elements or a combination of two or more
elements, unless the context clearly indicates otherwise. As used
herein, "comprises" means "includes." Thus, "comprising A or B,"
means "including A, B, or A and B," without excluding additional
elements. All Genbank Accession numbers are incorporated by
reference (the sequence available on Dec. 15, 2008).
[0049] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure
belongs.
[0050] HCC: hepatocellular carcinoma
[0051] IL-2: interleukin-2
[0052] MMP-9: matrix metalloproteinase-9
[0053] OPN: osteopontin
[0054] Administration: To provide or give a subject an agent, such
as a composition that includes a peptide fragment of OPN-5 kD, such
as one or more of SEQ ID NOS: 5-8, or OPN-5 kD specific antibodies,
by any effective route. Exemplary routes of administration include,
but are not limited to, oral, injection (such as subcutaneous,
intramuscular, intradermal, intraperitoneal, and intravenous),
sublingual, rectal, transdermal (e.g., topical), intranasal,
vaginal and inhalation routes.
[0055] Antibody: A polypeptide ligand comprising at least a light
chain or heavy chain immunoglobulin variable region which
specifically recognizes and binds an epitope of an antigen. For
example, OPN-5 kD specific antibodies include those that
specifically bind to the OPN-5 kD fragment antigen or an epitope
thereof (e.g., EELNGAY, amino acids 30 to 36 of SEQ ID NO: 4).
Antibodies are composed of a heavy and a light chain, each of which
has a variable region, termed the variable heavy (V.sub.H) region
and the variable light (V.sub.L) region. Together, the V.sub.H
region and the V.sub.L region are responsible for binding the
antigen recognized by the antibody. Includes polyclonal antibodies,
monoclonal antibodies, and fragments thereof.
[0056] This includes intact immunoglobulins and the variants and
portions of them well known in the art, such as Fab' fragments,
F(ab)'.sub.2 fragments, single chain Fv proteins ("scFv"), and
disulfide stabilized Fv proteins ("dsFv"). A scFv protein is a
fusion protein in which a light chain variable region of an
immunoglobulin and a heavy chain variable region of an
immunoglobulin are bound by a linker, while in dsFvs, the chains
have been mutated to introduce a disulfide bond to stabilize the
association of the chains. The term also includes genetically
engineered forms such as chimeric antibodies (for example,
humanized murine antibodies), heteroconjugate antibodies (such as,
bispecific antibodies). See also, Pierce Catalog and Handbook,
1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,
Immunology, 3.sup.rd Ed., W.H. Freeman & Co., New York,
1997.
[0057] Typically, a naturally occurring immunoglobulin has heavy
(H) chains and light (L) chains interconnected by disulfide bonds.
There are two types of light chain, lambda (.lamda.) and kappa (k).
There are five main heavy chain classes (or isotypes) which
determine the functional activity of an antibody molecule: IgM,
IgD, IgG, IgA and IgE.
[0058] Each heavy and light chain contains a constant region and a
variable region, (the regions are also known as "domains"). In
combination, the heavy and the light chain variable regions
specifically bind the antigen. Light and heavy chain variable
regions contain a "framework" region interrupted by three
hypervariable regions, also called "complementarity-determining
regions" or "CDRs". The extent of the framework region and CDRs
have been defined (see, Kabat et al., Sequences of Proteins of
Immunological Interest, U.S. Department of Health and Human
Services, 1991, which is hereby incorporated by reference). The
Kabat database is now maintained online. The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a species. The framework region of an antibody,
that is the combined framework regions of the constituent light and
heavy chains, serves to position and align the CDRs in
three-dimensional space.
[0059] The CDRs are primarily responsible for binding to an epitope
of an antigen. The CDRs of each chain are typically referred to as
CDR1, CDR2, and CDR3, numbered sequentially starting from the
N-terminus, and are also typically identified by the chain in which
the particular CDR is located. Thus, a V.sub.H CDR3 is located in
the variable domain of the heavy chain of the antibody in which it
is found, whereas a V.sub.L CDR1 is the CDR1 from the variable
domain of the light chain of the antibody in which it is found. An
antibody that binds an antigen of interest has a specific V.sub.H
region and the V.sub.L region sequence, and thus specific CDR
sequences. Antibodies with different specificities (due to
different combining sites for different antigens) have different
CDRs. Although it is the CDRs that vary from antibody to antibody,
only a limited number of amino acid positions within the CDRs are
directly involved in antigen binding. These positions within the
CDRs are called specificity determining residues (SDRs).
[0060] References to "V.sub.H" or "VH" refer to the variable region
of an immunoglobulin heavy chain, including that of an Fv, scFv,
dsFv or Fab. References to "V.sub.L" or "VL" refer to the variable
region of an immunoglobulin light chain, including that of an Fv,
scFv, dsFv or Fab.
[0061] A "monoclonal antibody" is an antibody produced by a single
clone of B-lymphocytes or by a cell into which the light and heavy
chain genes of a single antibody have been transfected, or a
progeny thereof. Monoclonal antibodies are produced by methods
known to those of skill in the art, for instance by making hybrid
antibody-forming cells from a fusion of myeloma cells with immune
spleen cells. Monoclonal antibodies include humanized monoclonal
antibodies. In some example a monoclonal antibody is the monoclonal
antibody 5 kd106-13 D, which is specific for the epitope EELNGAY
(amino acids 30 to 36 of SEQ ID NO: 4).
[0062] A "chimeric antibody" has framework residues from one
species, such as human, and CDRs or SDRs (which generally confer
antigen binding) from another species, such as a murine antibody
that specifically binds an epitope of the OPN-5 kD fragment. Most
typically, chimeric antibodies include human and murine antibody
domains, generally human constant regions and murine variable
regions, murine CDRs and/or murine SDRs. In some examples a
chimeric antibody includes the SDRs or CDRs from the monoclonal
antibody 5 kd106-13 D. In one example, a chimeric antibody is a
hybrid protein composed of the variable or antigen-binding domain
from a mouse antibody and the constant or effector domain from a
human antibody (such as an antibody that recognizes an epitope of
OPN-5 kD), although other mammalian species can be used, or the
variable region can be produced by molecular techniques. Methods of
making chimeric antibodies are well known in the art, for example,
see U.S. Pat. No. 5,807,715.
[0063] A "humanized antibody" is an immunoglobulin including a
human framework region and one or more CDRs from a non-human or
SDRs (for example a mouse, rat, or synthetic) immunoglobulin. The
non-human immunoglobulin providing the CDRs is termed a "donor,"
and the human immunoglobulin providing the framework is termed an
"acceptor." An exemplary donor is CDRs from monoclonal antibody 5
kd106-13 D. In one embodiment, all the CDRs are from the donor
immunoglobulin in a humanized immunoglobulin. Constant regions need
not be present, but if they are, they are substantially identical
to human immunoglobulin constant regions, such as at least about
85-90%, such as about 95% or more identical. Hence, all parts of a
humanized immunoglobulin, except possibly the CDRs, are
substantially identical to corresponding parts of natural human
immunoglobulin sequences. The acceptor framework of a humanized
immunoglobulin or antibody can have a limited number of
substitutions by amino acids taken from the donor framework.
Humanized or other monoclonal antibodies can have additional amino
acid substitutions which have substantially no effect on antigen
binding or other immunoglobulin functions. Exemplary conservative
substitutions are described above (see also U.S. Pat. No.
5,585,089). Humanized immunoglobulins can be constructed by means
of genetic engineering, for example, see U.S. Pat. Nos. 5,225,539
and 5,585,089.
[0064] Binding affinity: Affinity of an antibody for an antigen,
such as the affinity of an antibody (e.g., monoclonal antibody 5
kd106-13 D) for an OPN-5 kD peptide fragment or epitope thereof. In
one example, affinity is calculated by a modification of the
Scatchard method described by Frankel et al., Mol. Immunol.,
16:101-106, 1979. In another example, binding affinity is measured
by an antigen/antibody dissociation rate. In yet another example, a
high binding affinity is measured by a competition
radioimmunoassay. In several examples, a high binding affinity is
at least about 1.times.10.sup.-8 M. In other example, a high
binding affinity is at least about 1.5.times.10.sup.-8, at least
about 2.0.times.10.sup.-8, at least about 2.5.times.10.sup.-8, at
least about 3.0.times.10.sup.-8, at least about
3.5.times.10.sup.-8, at least about 4.0.times.10.sup.-8, at least
about 4.5.times.10.sup.-8, or at least about 5.0.times.10.sup.-8
M.
[0065] In a particular example, a functional variant of an OPN-5 kD
peptide retains a similar binding affinity for an antibody than the
binding affinity of the native OPN-5 kD peptide (such as SEQ ID NO:
4) for the same antibody.
[0066] Cancer: Malignant neoplasm that has undergone characteristic
anaplasia with loss of differentiation, increased rate of growth,
invasion of surrounding tissue, and is capable of metastasis.
[0067] CD44 receptors: A family of cell-surface adhesion molecules
found on both normal and malignant cell types that can mediate
cell-matrix and cell-cell interactions. CD44 receptors have been
associated with increased inflammation and metastasis. Based on the
results shown herein, it is proposed that OPN-5 kD is a ligand for
CD44 receptors, and this interaction enhances cellular invasion,
for example of HCC cells.
[0068] Chemotherapeutic agent: In cancer treatment, refers to the
administration of one or a combination of compounds to kill or slow
the reproduction of rapidly multiplying cells. Chemotherapeutic
agents include anti-neoplastics known by those skilled in the art,
including, but not limited to: 5-fluorouracil (5-FU), azathioprine,
cyclophosphamide, antimetabolites (such as Fludarabine),
antineoplastics (such as Etoposide, Doxorubicin, methotrexate, and
Vincristine), carboplatin, cis-platinum and the taxanes, such as
taxol, monoclonal antibodies such as Avastin or Herceptin, and
growth pathway inhibitors such as Gleevac. In particular examples,
such chemotherapeutic agents are administered in combination with a
therapy that reduces cellular invasion (for example before, during
or after administration of a therapeutic amount of one or more
fragments of OPN-5 kD that include at least 5 contiguous amino
acids of SEQ ID NO: 4, such as SEQ ID NOS: 5-8, or OPN-5 kD
specific antibodies).
[0069] Complementarity Determining Region (CDR): Amino acid
sequences which together define the binding affinity and
specificity of the natural Fv region of a native Ig binding site.
The light and heavy chains of an immunoglobulin each have three
CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3,
respectively. By definition, the CDRs of the light chain are
bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56
(L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are
bounded by the residues at positions 31 and 35b (H-CDR1), 50 and 65
(H-CDR2), 95 and 102 (H-CDR3), using the numbering convention
delineated by Kabat et al., (1991) Sequences of Proteins of
Immunological Interest, 5.sup.th Edition, U.S. Department of Health
and Human Services, Public Health Service, National Institutes of
Health, Bethesda, Md. (NIH Publication No. 91-3242). CDRs contain
the specificity determining regions (SDRs) of the antibody. In some
examples a CDR is a CDR from the monoclonal antibody 5 kd106-13
D.
[0070] Conservative substitution: One or more amino acid
substitutions for amino acid residues having similar biochemical
properties. Typically, conservative substitutions have little to no
impact on the activity of a resulting polypeptide. For example, a
conservative substitution in an OPN-5 kD fragment (such as one or
more conservative substitutions in any of SEQ ID NOS: 5-8) ideally
does not substantially affect the ability of the peptide to reduce
invasion of an HCC cell. Methods that can be used to determine the
amount of invasion are disclosed herein (for example, see Examples
4-8). In another particular example, a conservative substitution in
OPN-5 kD (such as SEQ ID NO: 4) ideally does not significantly
decrease the ability of OPN-5 kD to specifically bind to CD44, and
in some examples may not affect the ability of such an interaction
to stimulate cellular invasion. The interaction between an OPN-5 kD
variant containing one or more conservative amino acid
substitutions and CD44 can be measured using methods known in the
art, such as incubating a labeled OPN-5 kD antibody with CD44
receptor-expressing cells in the presence of OPN-5 kD, wherein the
presence of detectable label indicates the presence of bound OPN-5
kD to the cells.
[0071] For example, an alanine scan can be used to identify which
amino acid residues in SEQ ID NOS: 4-8 can tolerate an amino acid
substitution. In one example, one or more conservative
substitutions in SEQ ID NOS: 5-8 ideally do not decrease the
observed reduction of invasion by more than 25%, for example not
more than 20%, for example not more than 10%, when an alanine, or
other conservative amino acid (such as those listed below), is
substituted for one or more native amino acids. In one example, one
or more conservative substitutions in SEQ ID NO: 4 ideally does not
reduce the observed interaction of SEQ ID NO: 4 with CD44 receptor
by more than 25%, for example not more than 20%, for example not
more than 10%, when an alanine, or other conservative amino acid
(such as those listed below), is substituted for one or more native
amino acids.
[0072] In one example, one conservative substitution is included in
the peptide, such as a single conservative amino acid substitution
in any of SEQ ID NOS: 4-8. In another example, two conservative
substitutions are included in the peptide (such as any of SEQ ID
NOS: 4-8). In a further example, three conservative substitutions
are included in the peptide (such as any of SEQ ID NOS: 4-8). A
peptide can be produced to contain one or more conservative
substitutions by manipulating the nucleotide sequence that encodes
that polypeptide using, for example, standard procedures such as
site-directed mutagenesis or PCR. Alternatively, a peptide can be
produced to contain one or more conservative substitutions by using
standard peptide synthesis methods.
[0073] Substitutional variants are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Examples of amino acids which may be
substituted for an original amino acid in a protein and which are
regarded as conservative substitutions include: Ser for Ala; Lys
for Arg; Gln or His for Asn; Glu for Asp; Ser for Cys; Asn for Gln;
Asp for Glu; Pro for Gly; Asn or Gln for His; Leu or Val for Ile;
Ile or Val for Leu; Arg or Gln for Lys; Leu or Ile for Met; Met,
Leu or Tyr for Phe; Thr for Ser; Ser for Thr; Tyr for Trp; Trp or
Phe for Tyr; and Ile or Leu for Val.
[0074] Further information about conservative substitutions can be
found in, among other locations in, Ben-Bassat et al., (J.
Bacteriol. 169:751-7, 1987), O'Regan et al., (Gene 77:237-51,
1989), Sahin-Toth et al., (Protein Sci. 3:240-7, 1994), Hochuli et
al., (Bio/Technology 6:1321-5, 1988) and in standard textbooks of
genetics and molecular biology.
[0075] Decrease: To reduce the quality, amount, or strength of
something.
[0076] In one example, a therapy decreases a tumor (such as the
size or volume of a tumor, the number of tumors, the metastasis of
a tumor, or combinations thereof), or one or more symptoms
associated with a tumor, for example as compared to the response in
the absence of the therapy. In a particular example, a therapy
decreases the size or volume of a tumor, the number of tumors, the
metastasis of a tumor, or combinations thereof, subsequent to the
therapy, such as a decrease of at least 10%, at least 20%, at least
50%, or even at least 90%. Such decreases can be measured using the
methods disclosed herein.
[0077] Deletion: The removal of a one or more nucleotides from a
nucleic acid molecule or the removal of one or more amino acids
from a protein, the regions on either side being joined
together.
[0078] Effector molecule: The portion of a chimeric molecule, for
example a chimeric molecule that includes an antibody (e.g., 5
kd106-13 D) or fragment thereof, that is intended to have a desired
effect on a cell to which the chimeric molecule is targeted.
Effector molecules are also known as an effector moieties (EM),
therapeutic agents, or diagnostic agents, or similar terms.
[0079] Therapeutic agents include such compounds as nucleic acids,
toxins, proteins, peptides, amino acids or derivatives,
glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant
viruses. Nucleic acid therapeutic and diagnostic moieties include
antisense nucleic acids, derivatized oligonucleotides for covalent
cross-linking with single or duplex DNA, and triplex forming
oligonucleotides. Alternatively, the molecule linked to a targeting
moiety, such as an antibody, may be an encapsulation system, such
as a liposome or micelle that contains a therapeutic composition
such as a drug, a nucleic acid (such as an antisense nucleic acid),
or another therapeutic moiety that can be shielded from direct
exposure to the circulatory system. Means of preparing liposomes
attached to antibodies are well known to those of skill in the art.
See, for example, U.S. Pat. No. 4,957,735; and Connor et al.,
Pharm. Ther. 28:341-365, 1985. Diagnostic agents or moieties
include radioisotopes and other detectable labels. Detectable
labels useful for such purposes are also well known in the art, and
include radioactive isotopes such as .sup.32P, .sup.125I, and
.sup.131I, fluorophores, chemiluminescent agents, magnetic
resonance imaging agents and enzymes.
[0080] Epitope: An antigenic determinant. An epitope is the
particular structure formed by chemical groups or peptide sequences
in a molecule that is antigenic, meaning that elicits a specific
immune response. An antibody specifically binds a particular
antigenic epitope, for example on an OPN-5 kD peptide fragment or
portion thereof (e.g., EELNGAY, amino acids 30 to 36 of SEQ ID NO:
4). Epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of a
protein. Epitopes formed from contiguous amino acids are typically
retained on exposure to denaturing solvents whereas epitopes formed
by tertiary folding are typically lost on treatment with denaturing
solvents. An epitope typically includes at least 3, and more
usually, at least 5, 6, 7, 8, 9 or 10 amino acids in a unique
spatial conformation, such as this number of contiguous amino acids
from OPN-5 Kd (e.g., SEQ ID NO: 4). Methods of determining spatial
conformation of epitopes include, for example, x-ray
crystallography and 2-dimensional nuclear magnetic resonance. See,
for example, "Epitope Mapping Protocols" in Methods in Molecular
Biology, Vol. 66, Glenn E. Morris, Ed (1996).
[0081] Framework Region: Amino acid sequences interposed between
CDRs, and includes variable light and variable heavy framework
regions. The framework regions serve to hold the CDRs of an
antibody or fragment thereof in an appropriate orientation for
antigen binding.
[0082] Hepatocellular carcinoma (HCC): A malignant collection of
abnormal and uncontrollably growing cells that derive from
hepatocytes, the epithelial cells of the liver. Risk factors for
development of liver cancer include chronic infection with the
hepatitis B or C virus and cirrhosis (scarring of the liver). HCC
can be a primary tumor, an HCC tumor that has metastasized to
another part of the body, or can result from a metastasis to the
liver from another part of the body (such as from colon, stomach,
skin or ovarian cancer).
[0083] Immunoconjugate: A covalent linkage of an effector molecule
to an antibody, such as 5 kd106-13 D antibody. The effector
molecule can be a detectable label or a therapeutic molecule.
[0084] A "chimeric molecule" is a targeting moiety, such as a
ligand or an antibody, conjugated (coupled) to an effector
molecule. The term "conjugated" or "linked" refers to making two
polypeptides into one contiguous polypeptide molecule. In one
embodiment, an antibody is joined to an effector molecule (EM). In
another embodiment, an antibody joined to an effector molecule is
further joined to a lipid or other molecule to a protein or peptide
to increase its half-life in the body. The linkage can be either by
chemical or recombinant means. In one embodiment, the linkage is
chemical, wherein a reaction between the antibody moiety and the
effector molecule has produced a covalent bond formed between the
two molecules to form one molecule. A peptide linker (short peptide
sequence) can optionally be included between the antibody and the
effector molecule. Because immunoconjugates were originally
prepared from two molecules with separate functionalities, such as
an antibody and an effector molecule, they are also sometimes
referred to as "chimeric molecules."
[0085] Immunologically reactive conditions: Includes reference to
conditions which allow an antibody raised against a particular
epitope (e.g., EELNGAY, amino acids 30 to 36 of SEQ ID NO: 4) to
bind to that epitope (or cell expressing the epitope) to a
detectably greater degree than, and/or to the substantial exclusion
of, binding to substantially all other epitopes (or cells not
expressing the epitope). Immunologically reactive conditions are
dependent upon the format of the antibody binding reaction and
typically are those utilized in immunoassay protocols or those
conditions encountered in vivo. See Harlow & Lane (Antibodies:
A Laboratory Manual. 1988) for a description of immunoassay formats
and conditions. The immunologically reactive conditions employed in
the methods are "physiological conditions" which include reference
to conditions (such as temperature, osmolarity, pH) that are
typical inside a living mammal or a mammalian cell. While it is
recognized that some organs are subject to extreme conditions, the
intra-organismal and intracellular environment normally lies around
pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5),
contains water as the predominant solvent, and exists at a
temperature above 0.degree. C. and below 50.degree. C. Osmolarity
is within the range that is supportive of cell viability and
proliferation.
[0086] Isolated: An "isolated" biological component (such as a
nucleic acid molecule, protein, antibody, or cell) has been
substantially separated or purified away from other components,
such as other components in the cell of the organism, or the
organism itself, in which the component occurs, such as other
chromosomal and extra-chromosomal DNA and RNA, proteins and cells.
Nucleic acid molecules and proteins that have been "isolated"
include nucleic acid molecules (such as DNA or RNA) and proteins
purified by standard purification methods. The term also embraces
nucleic acid molecules and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acid molecules and proteins. For example, an isolated antibody is
one that is substantially separated from the animal or cells in
which it was generated, or from cell culture medium.
[0087] Label: A detectable compound or composition that is
conjugated directly or indirectly to another molecule, such as an
antibody (e.g., 5 kd106-13 D) or a protein, to facilitate detection
of that molecule. For example, the label can be capable of
detection by ELISA, spectrophotometry, flow cytometry, or
microscopy. Specific, non-limiting examples of labels include
fluorophores, chemiluminescent agents, enzymatic linkages,
electron-dense compounds, haptens and radioactive isotopes. Methods
for labeling and guidance in the choice of labels appropriate for
various purposes are discussed for example in Sambrook et al.
(Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.,
1989) and Ausubel et al. (In Current Protocols in Molecular
Biology, John Wiley & Sons, New York, 1998).
[0088] Linker peptide: A peptide within an antibody binding
fragment (such as an Fv fragment, for example a 5 kd106-13 D
antibody fragment) which serves to indirectly bond the variable
heavy chain to the variable light chain. "Linker" can also refer to
a peptide serving to link a targeting moiety, such as a scFv, to an
effector molecule, such as a cytotoxin or a detectable label.
[0089] The terms "conjugating," "joining," "bonding" or "linking"
refer to making two polypeptides into one contiguous polypeptide
molecule, or to covalently attaching a radionuclide or other
molecule to a polypeptide, such as an scFv. In the specific
context, the terms include reference to joining a ligand, such as
an antibody moiety, to an effector molecule ("EM"). The linkage can
be either by chemical or recombinant means. "Chemical means" refers
to a reaction between the antibody moiety and the effector molecule
such that there is a covalent bond formed between the two molecules
to form one molecule.
[0090] Malignant: Cells which have the properties of anaplasia
invasion and metastasis.
[0091] Matrix metalloproteinase-9 (MMP-9): A member of the
metalloproteinase family, also referred to as gelatinase B. It is
called a gelatinase because it has a high affinity for digestion of
denatured collagen I. MMP-9 can also cleave a variety of proteins
including many components of the extracellular matrix such as
collagens I, III, IV, and V, entactin, and elastin. MMP-9 is
frequently up-regulated in cancer cells and also in the adjacent
host tissues, and its expression by tumor cells contributes to
metastasis.
[0092] MMP-9 has a signal peptide that leads to its secretion, but
the secreted molecule is not itself enzymatically active. MMP-9 has
a proregion adjacent to the signal peptide. The proregion contains
a signature sequence including an unpaired cysteine that interacts
with the Zn.sup.2+ that is complexed with three histidines to form
the active site. This interaction allows the proregion to act as a
competitive inhibitor of MMP-9. Hence, the proregion is cleaved and
dissociated to allow enzymatic activity
[0093] MMP-9 has been cloned from a variety of organisms, and
nucleic acid and protein sequences are publicly available, for
example from GenBank and EMBL (for example GenBank Accession Nos.
CAC07541.1; NP.sub.--038627.1; P14780.2; and NM.sub.--004994.2).
Human MMP-9 protein is Mr 92,000 although the murine form is Mr
105,000 because of an insert of an additional 24 amino acids.
[0094] Neoplasm: Abnormal growth of cells.
[0095] Normal cells: Non-tumor, non-malignant cells.
[0096] Osteopontin (OPN, SPP1): A secreted multi-functional
phosphorylated glycoprotein expressed at high levels in tumors and
the surrounding stroma of numerous cancers, including those of the
liver. OPN proteins contain a functional Gly-Arg-Gly-Asp-Ser
(GRGDS; amino acids 158-162 of SEQ ID NO: 2) cell-binding
sequence.
[0097] Several splice variants of OPN have been identified,
including OPN-a (native sequence) and OPN-c (truncated sequence).
OPN-c lacks exon 4 (27 amino acids) in the NH.sub.2-terminal region
of the mature sequence. OPN-c includes a transglutaminase reactive
domain (Gly-X-Gly) which can mediate covalent homodimer
cross-linking as well as heterodimer formation to other matrix
components (such as fibronectin).
[0098] The term osteopontin includes any osteopontin gene, cDNA,
mRNA, or protein from any organism that retains OPN biological
activity. OPN sequences are publicly available. For example,
GenBank Accession Nos: D28759 (nucleic acid) and BAA05949 (protein)
disclose human OPN-a sequences, and GenBank Accession Nos: D2876
(nucleic acid) and BAA05951 (protein) disclose human OPN-c
sequences.
[0099] In certain examples, OPN has at least 80% sequence identity,
for example at least 85%, at least 90%, at least 95%, or at least
98% sequence identity to a native OPN and retain OPN biological
activity. In other examples, an OPN nucleic acid sequence has a
sequence that hybridizes under very high stringency conditions to a
sequence set forth in GenBank Accession No. D28759 or D2876 and
retains OPN activity.
[0100] OPN-5 kD or OPN-5 kD fragment: One of three osteopontin
peptide fragments generated when exposed to MMP-9 (when run on a
SDS gel this peptide can migrate at approximately 10 kD, however,
its predicted size based upon amino acid sequence is 5 kD). This
peptide binds to the CD44 receptor, thereby enhancing cellular
invasion of HCC cells. A particular example of an OPN-5 kD sequence
is provided in SEQ ID NO: 4. Methods of diagnosing a tumor (such as
an OPN-c overexpressing tumor) by detecting OPN-5 kD fragment, are
provided herein. Peptide fragments of OPN-5 kD are provided herein,
and can be used for example to treat an OPN-overexpressing tumor or
to generate antibodies. Peptide fragments of OPN-5 kD include at
least 5 contiguous amino acids of SEQ ID NO: 4). Specific examples
of peptide fragments of OPN-5 kD include peptides of 5 to 60 or 5
to 50 amino acids in length (such as 5 to 25, 5 to 20, 5 to 15, 6
to 12, 8 to 10, 10 to 44, 10 to 20, or 10 to 15 amino acids) and
have least 5 contiguous amino acids of an OPN-5 kD sequence (e.g.,
SEQ ID NO: 4), such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 15, 16,
17, 18, 19, or 20 contiguous amino acids, such as 5 to 15 or 10 to
15 contiguous amino acids. Particular examples of peptide fragments
of OPN5-kD are shown in SEQ ID NOS: 5-8 and amino acids 30-36 of
SEQ ID NO: 4.
[0101] Peptide: A chain of amino acids of which is at least 4 amino
acids in length, regardless of post-translational modification
(such as glycosylation or phosphorylation). In one example, a
peptide is at least 6 amino acids in length, such as at least 8, at
least 9, at least 10, at least 11, or at least 12 amino acids in
length. In particular examples, a peptide is 4 to 30 amino acids in
length, for example 5 to 25 amino acids in length, 5 to 20 amino
acids in length, 9 to 15 amino acids in length, or 9 to 10 amino
acids in length.
[0102] Pharmaceutically Acceptable Carriers: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic agents, such as one or more compositions
that include (1) a peptide fragment of OPN-5 kD or (2) an OPN-5 kD
specific antibody (e.g., 5 kd106-13 D).
[0103] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations can include injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. In addition to
biologically-neutral carriers, pharmaceutical compositions to be
administered can contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
and pH buffering agents and the like, for example sodium acetate or
sorbitan monolaurate, sodium lactate, potassium chloride, calcium
chloride, and triethanolamine oleate.
[0104] Promoter: An array of nucleic acid control sequences which
direct transcription of a nucleic acid. A promoter includes
necessary nucleic acid sequences near the start site of
transcription, such as, in the case of a polymerase II type
promoter, a TATA element. A promoter also optionally includes
distal enhancer or repressor elements which can be located as much
as several thousand base pairs from the start site of
transcription. Promoters may be constitutive or inducible.
[0105] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified peptide is one in which the peptide is more
pure than the peptide in its natural environment, such as within a
cell.
[0106] In particular examples, purified OPN peptides or fragments
thereof (such as SEQ ID NOS: 4-8) refers to peptides that are at
least 75% pure, at least 80% pure, at least 90% pure, at least 95%
pure, at least 97% pure, at least 98% pure, or at least 99% pure.
The purity of a peptide can be measured using methods known in the
art, such as by a Western blot.
[0107] Radiological agent: In cancer treatment, refers to the
administration of one or a combination of radioactive compounds to
damage the DNA of cells, thereby killing or slowing the
reproduction of rapidly multiplying cells. Exemplary methods of
administering radiological agents to a subject include external
beam radiotherapy (XBRT) or teletherapy, brachytherapy or sealed
source radiotherapy, and unsealed source radiotherapy. The
radiological agents that can be administered to a subject in
combination with the disclosed therapies that include a peptide of
5 to 50 amino acids in length that includes at least 5 contiguous
amino acids of OPN-5 kD (such as at least 9 contiguous amino acids
of SEQ ID NO: 4) or OPN-5 kD antibodies. Exemplary radiological
agents include those known by those skilled in the art, such as
ionizing radiation (for example x-rays and gamma rays).
[0108] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence. This artificial combination can for example,
be accomplished by chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acids, such as by
genetic engineering techniques. Similarly, a recombinant protein is
one encoded for by a recombinant nucleic acid molecule.
[0109] Sample: Includes biological samples that contain cells,
genomic DNA, RNA, or proteins (or combinations thereof) obtained
from a subject, such as those present in peripheral blood (or a
fraction thereof such as plasma or serum), urine, saliva, tissue
biopsy, surgical specimen, fine needle aspirate, and autopsy
material. In a particular example, a sample is obtained from a
subject having or suspected of having a metastatic HCC.
[0110] Sequence identity: The identity between two or more nucleic
acid sequences, or two or more amino acid sequences, is expressed
in terms of the identity or similarity between the sequences.
Sequence identity can be measured in terms of percentage identity;
the higher the percentage, the more identical the sequences are.
Sequence similarity can be measured in terms of percentage
similarity (which takes into account conservative amino acid
substitutions); the higher the percentage, the more similar the
sequences are. Homologs or orthologs of nucleic acid or amino acid
sequences possess a relatively high degree of sequence
identity/similarity when aligned using standard methods.
[0111] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith & Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman & Wunsch, J. Mol. Biol. 48:443, 1970; Pearson &
Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins &
Sharp, Gene, 73:237-44, 1988; Higgins & Sharp, CABIOS 5:151-3,
1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988; Huang et
al. Computer Appls. in the Biosciences 8, 155-65, 1992; and Pearson
et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J. Mol.
Biol. 215:403-10, 1990, presents a detailed consideration of
sequence alignment methods and homology calculations.
[0112] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403-10, 1990) is available from several
sources, including the National Center for Biological Information
(NCBI, National Library of Medicine, Building 38A, Room 8N805,
Bethesda, Md. 20894) and on the Internet, for use in connection
with the sequence analysis programs blastp, blastn, blastx, tblastn
and tblastx. Additional information can be found at the NCBI web
site.
[0113] BLASTN is used to compare nucleic acid sequences, while
BLASTP is used to compare amino acid sequences. To compare two
nucleic acid sequences, the options can be set as follows: -i is
set to a file containing the first nucleic acid sequence to be
compared (such as C:\seq1.txt); -j is set to a file containing the
second nucleic acid sequence to be compared (such as C:\seq2.txt);
-p is set to blastn; -o is set to any desired file name (such as
C:\output.txt); -q is set to -1; -r is set to 2; and all other
options are left at their default setting. For example, the
following command can be used to generate an output file containing
a comparison between two sequences: C:\B12seq c:\seq1.txt -j
c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2.
[0114] To compare two amino acid sequences, the options of B12seq
can be set as follows: -i is set to a file containing the first
amino acid sequence to be compared (such as C:\seq1.txt); -j is set
to a file containing the second amino acid sequence to be compared
(such as C:\seq2.txt); -p is set to blastp; -o is set to any
desired file name (such as C:\output.txt); and all other options
are left at their default setting. For example, the following
command can be used to generate an output file containing a
comparison between two amino acid sequences: C:\B12seq c:\seq1.txt
-j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared
sequences share homology, then the designated output file will
present those regions of homology as aligned sequences. If the two
compared sequences do not share homology, then the designated
output file will not present aligned sequences.
[0115] Once aligned, the number of matches is determined by
counting the number of positions where an identical nucleotide or
amino acid residue is presented in both sequences. The percent
sequence identity is determined by dividing the number of matches
either by the length of the sequence set forth in the identified
sequence, or by an articulated length (such as 100 consecutive
nucleotides or amino acid residues from a sequence set forth in an
identified sequence), followed by multiplying the resulting value
by 100. For example, a nucleic acid sequence that has 1166 matches
when aligned with a test sequence having 1154 nucleotides is 75.0
percent identical to the test sequence (1166=1554*100=75.0). The
percent sequence identity value is rounded to the nearest tenth.
For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to
75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to
75.2. The length value will always be an integer. In another
example, a target sequence containing a 20-nucleotide region that
aligns with 20 consecutive nucleotides from an identified sequence
as follows contains a region that shares 75 percent sequence
identity to that identified sequence (that is, 15/20*100=75).
[0116] For comparisons of amino acid sequences of greater than
about 30 amino acids, the Blast 2 sequences function is employed
using the default BLOSUM62 matrix set to default parameters, (gap
existence cost of 11, and a per residue gap cost of 1). Homologs
are typically characterized by possession of at least 70% sequence
identity counted over the full-length alignment with an amino acid
sequence using the NCBI Basic Blast 2.0, gapped blastp with
databases such as the nr or swissprot database. Queries searched
with the blastn program are filtered with DUST (Hancock and
Armstrong, 1994, Comput. Appl. Biosci. 10:67-70). Other programs
use SEG. In addition, a manual alignment can be performed. Proteins
with even greater similarity will show increasing percentage
identities when assessed by this method, such as at least 75%, at
least 80%, at least 85%, at least 90%, at least 95%, at least 98%,
or at least 99% sequence identity. For example, a peptide having
substantial sequence identity to an OPN-5 kD sequence can share at
least 80% sequence identity to SEQ ID NO: 4, such as at least 90%
or at least 95% sequence identity to SEQ ID NO: 4.
[0117] When aligning short peptides (fewer than around 30 amino
acids), the alignment can be performed using the Blast 2 sequences
function, employing the PAM30 matrix set to default parameters
(open gap 9, extension gap 1 penalties). For example, when less
than the entire sequence is being compared for sequence identity,
homologs will typically possess at least 75% sequence identity over
short windows of 10-20 amino acids, and can possess sequence
identities of at least 85%, at least 90%, at least 95% or at least
98% depending on their identity to the reference sequence. For
example, a peptide having substantial sequence identity to an OPN-5
kD fragment can share at least 80% sequence identity to any of SEQ
ID NOS: 5-8, such as at least 90%, at least 95%, at least 98%, or
at least 99% sequence identity to SEQ ID NOS: 5-8. Methods for
determining sequence identity over such short windows are described
at the NCBI web site.
[0118] One indication that two nucleic acid molecules are closely
related is that the two molecules hybridize to each other under
stringent conditions, as described above. Nucleic acid sequences
that do not show a high degree of identity may nevertheless encode
identical or similar (conserved) amino acid sequences, due to the
degeneracy of the genetic code. Changes in a nucleic acid sequence
can be made using this degeneracy to produce multiple nucleic acid
molecules that all encode substantially the same protein. Such
homologous nucleic acid sequences can, for example, possess at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 98%, or at least 99% sequence identity determined by this
method. For example, a nucleic acid encoding an OPN-5 kD sequence
can have substantial sequence identity to a native sequence if it
shares at least 80% sequence identity to nucleotides 566-697 of SEQ
ID NO: 1, such as at least 90% or at least 95% sequence identity to
nucleotides 566-697 of SEQ ID NO: 1. An alternative (and not
necessarily cumulative) indication that two nucleic acid sequences
are substantially identical is that the peptide which the first
nucleic acid sequence encodes is immunologically cross reactive
with the peptide encoded by the second nucleic acid sequence.
[0119] One of skill in the art will appreciate that the particular
sequence identity ranges are provided for guidance only; it is
possible that strongly significant homologs could be obtained that
fall outside the ranges provided.
[0120] Specific binding agent: An agent that binds substantially
only to a defined target. Thus an OPN-5 kD specific binding agent
is an agent that binds substantially to a OPN-5 kD peptide or
epitope thereof. In one example, the specific binding agent is an
antibody that specifically binds the OPN-5 kD peptide.
[0121] The term "specifically binds" refers, with respect to an
antigen such as OPN-5 kD, to the preferential association of an
antibody or other specific binding agent, in whole or part, to the
antigen and not to other antigens. A certain degree of non-specific
interaction can occur between a specific binding agent and a
non-target antigen. Nevertheless, specific binding can be
distinguished as mediated through specific recognition of the
antigen. Specific binding results in a significant association
between the antibody (or other specific binding agent) and the
antigen than between the antibody and a non-antigen. Specific
binding typically results in greater than 2-fold, such as greater
than 5-fold, greater than 10-fold, or greater than 100-fold
increase in amount of bound antibody or other specific binding
agent to the antigen as compared to binding to a non-specific
antigen.
[0122] The determination that a particular agent binds
substantially only to OPN-5 kD can be made using or adapting
routine procedures. For example, western blotting can be used to
determine that a specific binding agent, such as an antibody, binds
substantially only to the protein (such as substantially only binds
OPN-5 kD but not to other proteins, such as those found in blood)
(for example see Harlow and Lane, Antibodies: A Laboratory Manual.
1988). A variety of immunoassay formats are appropriate for
selecting antibodies or other specific binding agent specifically
immunoreactive with a particular protein (such as OPN-5 kD). For
example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein.
[0123] Subject: Living multi-cellular vertebrate organisms, a
category that includes human and non-human mammals (such as
laboratory or veterinary subjects).
[0124] Therapeutically effective amount: An amount of a therapeutic
agent (such as an OPN-5 kD specific antibody, or an OPN-5 kD
peptide fragment (such as a peptide of 5 to 50 amino acids in
length that includes at least 5 contiguous amino acids of OPN-5
kD), that alone, or together with one or more additional
therapeutic agents, induces the desired response, such as treatment
of a tumor that overexpresses OPN, such as a metastatic HCC tumor.
In one example, it is an amount of an OPN-5 kD specific antibody,
or one or more peptide fragments of OPN-5 kD needed to prevent or
delay the development of a tumor, prevent or delay the metastasis
of a tumor, cause regression of an existing tumor, or treat one or
more signs or symptoms associated with a tumor, in a subject, such
as a subject having HCC. Ideally, a therapeutically effective
amount provides a therapeutic effect without causing a substantial
cytotoxic effect in the subject. The preparations disclosed herein
are administered in therapeutically effective amounts.
[0125] In one example, a desired response is to decrease the size,
volume, or number (such as metastases) of a tumor that
overexpresses OPN, such as HCC. For example, the therapeutic
compositions can decrease the size, volume, or number of tumors
(such as HCC) by a desired amount, for example by at least 5%, at
least 10%, at least 15%, at least 20%, at least 25%, at least 30%,
at least 50%, at least 75%, or even at least 90%, as compared to a
response in the absence of the composition.
[0126] The effective amount of a composition that includes an OPN-5
kD specific antibody, or one or more peptide fragments of OPN-5 kD,
that is administered to a human or veterinary subject will vary
depending upon a number of factors associated with that subject,
for example the overall health of the subject. An effective amount
of a composition can be determined by varying the dosage of the
product and measuring the resulting therapeutic response, such as
the regression of a tumor. Effective amounts also can be determined
through various in vitro, in vivo or in situ immunoassays. The
disclosed therapeutic agents can be administered in a single dose,
or in several doses, as needed to obtain the desired response.
However, the effective amount of can be dependent on the source
applied, the subject being treated, the severity and type of the
condition being treated, and the manner of administration.
[0127] In particular examples, a therapeutically effective dose of
one or more peptide fragments of OPN-5 kD or an OPN-5 kD antibody
includes at least 1 .mu.g daily (such as 1-100 .mu.g or 5-50 .mu.g)
if administered via injection, or at least 1 mg daily if
administered topically (such as 1-100 mg or 5-50 mg) of one or more
peptides fragments of OPN-5 kD or an OPN-5 kD antibody. In
particular examples, such daily dosages are administered in one or
more divided doses (such as 2, 3, or 4 doses) or in a single
formulation.
[0128] The disclosed compositions that include one or more peptide
fragments of OPN-5 kD or an OPN-5 kD antibody can be administered
alone, in the presence of a pharmaceutically acceptable carrier, in
the presence of other therapeutic agents (such as other
anti-neoplastic agents), or both.
[0129] Transgene: An exogenous gene introduced into a cell.
[0130] Treating or treatment: Refers to a therapeutic intervention
that ameliorates a sign or symptom of a disease or pathological
condition related to a disease (such as a tumor, for example HCC).
Treatment can also induce remission or cure of a condition, such as
a tumor. In particular examples, treatment includes preventing a
tumor, for example by inhibiting the full development of a tumor,
such as preventing development of a metastasis or the development
of a primary tumor. Prevention does not require a total absence of
a tumor.
[0131] Reducing a sign or symptom associated with a tumor (such as
a tumor that overexpresses OPN, for example HCC) can be evidenced,
for example, by a delayed onset of clinical symptoms of the disease
in a susceptible subject (such as a subject having HCC which has
not yet metastasized), a reduction in severity of some or all
clinical symptoms of the disease, a slower progression of the
disease (for example by prolonging the life of a subject having
tumor), a reduction in the number of relapses of the disease, an
improvement in the overall health or well-being of the subject, or
by other parameters well known in the art that are specific to the
particular tumor.
[0132] Tumor: A neoplasm.
[0133] Under conditions sufficient for: A phrase that is used to
describe any environment that permits the desired activity.
[0134] In one example, includes administering a therapeutically
effective amount of a composition that includes an OPN-5 kD
specific antibody or one or more peptides fragments of OPN-5 kD
sufficient to allow the desired activity. In particular examples
the desired activity is treatment of a tumor, such as a tumor that
expresses OPN, for example HCC.
[0135] Unit dose: A physically discrete unit containing a
predetermined quantity of an active material calculated to
individually or collectively produce a desired effect, such as a
therapeutic effect. A single unit dose or a plurality of unit doses
can be used to provide the desired effect, such as treatment of a
tumor, for example a metastatic HCC. In one example, a unit dose
includes a desired amount of an agent that decreases cellular
invasion induced by OPN-5 kD binding to CD44.
[0136] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector can
include nucleic acid sequences that permit it to replicate in the
host cell, such as an origin of replication. A vector may also
include one or more therapeutic genes and/or selectable marker
genes and other genetic elements known in the art. A vector can
transduce, transform or infect a cell, thereby causing the cell to
express nucleic acids and/or proteins other than those native to
the cell. A vector optionally includes materials to aid in
achieving entry of the nucleic acid into the cell, such as a viral
particle, liposome, protein coating or the like. In a particular
example, a vector includes a nucleic acid molecule encoding one or
more of SEQ ID NOS: 4-8 or a fragment of SEQ ID NO: 4.
Methods of Treating a Tumor
[0137] It is shown herein that MMP-9 liberates an OPN-5 kD
fragment, and that OPN-induced cellular invasion proceeds via
binding of the OPN-5 kD ligand to CD44 cell surface receptors.
Without being bound to a particular theory, it is proposed that
exposure of a non-RGD cryptic binding site region of OPN by
extracellular proteolytic cleavage increases binding to cell
surface CD44 receptors and mediates an invasive response, which is
distinct and independent from integrin .alpha.V.beta.3 function. It
is also demonstrated herein that small peptide fragments of OPN-5
kD (for example any of SEQ ID NOS: 5-8) effectively decrease or
inhibit cellular invasion induced by OPN-5 kD. Without being bound
to a particular theory, it is proposed that these peptides
structurally compete with OPN-5 kD for available cell-surface CD44
docking sites, thereby decreasing cellular invasion. This
observation also supports the use of antibodies directed to
epitopes within the OPN-5 kD peptide (such as amino acids 30-36 of
SEQ ID NO: 4) as therapeutics. Based on these observations, methods
of treating a tumor that overexpresses OPN, MMP-9, or both, are
disclosed. These methods include administering peptides,
antibodies, or combinations thereof, which effectively decrease or
inhibit cellular invasion.
[0138] A particular example of an OPN-5 kD sequence is provided in
SEQ ID NO: 4. However, one skilled in the art will appreciate that
variants of SEQ ID NO: 4 (such as allelic variations that may be
present between individuals or organisms, which may include one or
more substitutions, deletions, or insertions, or combinations
thereof) can also be an OPN-5 kD peptide, as long as such peptide
retains the ability to bind CD44 receptor an stimulate cellular
invasion of HCC cells (for example using the methods described in
Examples 4-7). In a particular example, an OPN-5 kD sequence is 40
to 50 amino acids in length (such as 40-48, 42-46, or 44 amino
acids), and has at least 90% sequence identity to SEQ ID NO: 4,
such as at least 92%, at least 95%, or at least 98% sequence
identity to SEQ ID NO: 4. In one example, an OPN-5 kD peptide is 40
to 50 amino acids in length and includes 1-8 conservative amino
acid substitutions in SEQ ID NO: 4, such as 1, 2, 3, 4, 5, 6, 7 or
8 conservative amino acid substitutions in SEQ ID NO: 4.
[0139] Methods are disclosed herein for treating tumors, such as
those that overexpress OPN, for example in combination with
overexpression of MMP-9. In one example, increased expression of
OPN or MMP-9 can be detected in serum or plasma obtained from a
subject having such a tumor. For example, detection of OPN-5 kD in
the serum of a subject (for example at a level of at least twice
that found in a subject not having a tumor), detection of increased
levels of OPN-c in the tumor (for example relative to expression of
OPN-c in adjacent non-tumor cells, or relative to expression of
OPN-a in the tumor), or both, indicates that the subject can
benefit from the disclosed therapies. In some examples, subjects
are initially screened to determine if they have increased levels
of OPN-5 kD in their serum, whether they have a tumor that has
increased expression of OPN-c (for example relative to adjacent
non-tumor cells, or relative to expression of OPN-a), or
combinations thereof. For example, the diagnostic methods provided
herein can be used to screen subjects to determine if they are
candidates for the disclosed therapies.
[0140] In some examples, the tumor is treated in vivo, for example
in a mammalian subject, such as a human or veterinary subject. A
tumor is an abnormal growth of tissue that results from excessive
cell division. A particular example of a tumor is cancer. For
example, the current application is useful for the treatment (such
as the prevention or reduction of metastasis) of tumors (such as
cancers). Exemplary tumors that can be treated using the disclosed
methods include, but are not limited to: cancers of the liver,
breast, colon, and prostate, including metastases of such tumors.
For example, the tumor can be a tumor that overexpresses OPN (such
as OPN-c), for example HCC, metastatic HCC, such as an
intra-hepatic metastasis or an extra-hepatic metastasis.
[0141] Treatment of a tumor, such as HCC or metastatic HCC, can
include preventing or delaying the development of the tumor in a
subject (such as preventing metastasis of a tumor), and also
includes reducing signs or symptoms associated with the presence of
such a tumor (for example by reducing the size or volume of the
tumor or a metastasis thereof). In a specific example, treatment
includes reducing the growth of cells of the tumor, or even killing
the tumor cells (for example by causing the cells to undergo
apoptosis). Such reduced growth can in some examples decrease or
slow metastasis of the tumor, or reduce the size or volume of the
tumor. In one example, treatment of a tumor includes reducing the
invasive activity of the tumor in the subject, for example by
reducing the ability of the tumor to metastasize. In some examples,
treatment using the methods disclosed herein prolongs the time of
survival of the subject (e.g., increases survival time by at least
6 months, at least 9 months, at least 12 months, at least 2 years,
at least 3 years, or even at least 5 years relative to the absence
of the therapy).
[0142] In particular examples, the method includes administering to
the subject a therapeutically effective amount of one or more
agents that reduce cellular invasion resulting from the interaction
between OPN-5 kD and CD44 receptor, thereby treating the tumor. In
particular examples, cellular invasion is reduced by at least 10%
(such as at least 20%, at least 50%, or at least 90%), for example
as compared to an amount of cellular invasion in the absence of the
therapeutic agent. Examples of such agents include peptide
fragments of OPN-5 kD or antibodies specific to OPN-5 kD (e.g., 5
kd106-13 D) that reduce the interaction between OPN-5 kD and CD44
receptor and reduce cellular invasion. Further detail on the OPN-5
kD antibodies that can be used therapeutically is provided
below.
[0143] Particular examples of agents that can be used in the
methods disclosed herein include therapeutic amounts of peptide
fragments of OPN-5 kD. Such fragments include peptides of 5 to 50
amino acids (such as 5 to 25, 10 to 44, 10 to 20, or 10 to 15 amino
acids) that include at least 5 contiguous amino acids of an OPN-5
kD sequence (e.g., SEQ ID NO: 4), such as at least 6, at least 7,
at least 8, at least 8, at least 9, at least 10 or at least 15
contiguous amino acids of SEQ ID NO: 4, for example 5-20, 10-15, or
8-10 contiguous amino acids of SEQ ID NO: 4. For example, the
therapeutic peptide can include or consist of 5, 6, 7, 8, 9, 10,
12, 15, 20, or 30 contiguous amino acids of SEQ ID NO: 4. In one
example, the peptide fragment of OPN-5 kD include or consists of
the amino acid sequence of SEQ ID NO: 5, 6, 7, or 8 or amino acids
30-36 of SEQ ID NO: 4. One skilled in the art will appreciate that
more than one peptide fragment of OPN-5 kD can be used, such as one
or more peptides consisting of SEQ ID NO: 5, 6, 7, or 8. In some
examples the therapeutic peptide is a fusion peptide, such as a
fusion peptide that includes any of SEQ ID NOS: 5-8.
Screening Subjects
[0144] Subjects that can benefit from the disclosed therapies
include human and veterinary subjects. Subjects can be screened
prior to initiating the disclosed therapies, for example to
determine whether the subject has a tumor that overexpresses OPN,
MMP-9, or both. The presence of a tumor that overexpresses OPN,
MMP-9, or both indicates that the tumor can be treated using the
methods provided herein.
[0145] In one example, the tumor (or a portion thereof, such as a
fine needle aspirate or other biopsy sample) is analyzed using
immunodetection methods. For example, the biological sample can be
incubated with an antibody that specifically binds to OPN or to
MMP-9, or both antibodies. The primary antibody can include a
detectable label. For example, the primary antibody can be directly
labeled, or the sample can be subsequently incubated with a
secondary antibody that is labeled (for example with a fluorescent
label). The label can then be detected, for example by microscopy,
ELISA, flow cytometery, or spectrophotometry. In another example,
the biological sample is analyzed by Western blotting for the
presence of OPN or to MMP-9. In one example, a subject is screened
by determining whether increased levels of the OPN-5 kD fragment is
present in their serum (for example relative to a level present in
a serum sample from a subject not having a tumor), for example
using an antibody that specifically binds OPN-5 kD (such as those
described in Example 9).
[0146] As an alternative to analyzing the sample for the presence
of proteins, the presence of nucleic acids can be determined. For
example, the biological sample can be incubated with primers that
permit the amplification of OPN or to MMP-9, under conditions
sufficient to permit amplification of OPN or to MMP-9. Exemplary
methods include PCR and RT-PCR. In another example, the biological
sample is incubated with probes that can bind to OPN or to MMP-9
nucleic acid (such as cDNA, genomic DNA, or RNA (such as mRNA))
under high stringency conditions. The resulting hybridization can
then be detected using methods known in the art. In one example, a
subject is screened by determining whether increased levels of
OPN-c are present in the tumor (for example relative to a level
present in adjacent non-tumor cells from the same subject, or
relative to a level of OPN-a in the same tumor sample), for example
detecting OPN-c (and in some examples also OPN-a) mRNA
expression.
[0147] The presence of increased expression of OPN or MMP-9
indicates that the tumor overexpresses OPN or MMP-9 or both, and
that the tumor is one that can be treated using the disclosed
therapies. In one example, the presence of increased OPN-5 kD in
the serum, the presence of increased OPN-c in the tumor, or both,
indicates that the subject has a tumor that can be treated using
the disclosed therapies.
Administration
[0148] Methods of administration of the disclosed therapeutic
agents are routine, and can be determined by a skilled clinician.
For example, the disclosed therapies (such as peptide fragments of
OPN-5 kD and antibodies directed to an epitope of OPN-5 kD) can be
administered via injection (for example via embolization), orally,
topically, transdermally, parenterally, or via inhalation or spray.
In a particular example, a peptide consisting of 5 to 20 contiguous
amino acids of OPN-5 kD or an antibody specific for OPN-5 kD (such
as 5 kd106-13 D) is administered intravenously to a mammalian
subject, such as a human.
[0149] The therapeutic compositions, such as those that include
peptide fragments of OPN-5 kD or antibodies specific for OPN-5 kD,
can further include one or more biologically active or inactive
compounds (or both), such as anti-neoplastic agents and
conventional non-toxic pharmaceutically acceptable carriers,
respectively.
[0150] In a particular example, a therapeutic composition that
includes a therapeutically effective amount of one or more agents
that reduce cellular invasion due to the interaction of OPN-5 kD
with CD44 (such as peptide fragments of OPN-5 kD or antibodies
specific for OPN-5 kD) further includes one or more biologically
inactive compounds. Examples of such biologically inactive
compounds include, but are not limited to: carriers, thickeners,
diluents, buffers, preservatives, and carriers. The
pharmaceutically acceptable carriers useful for these formulations
are conventional (see Remington's Pharmaceutical Sciences, by E. W.
Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995)). In
general, the nature of the carrier will depend on the particular
mode of administration being employed. For instance, parenteral
formulations can include injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can include minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0151] In a particular example, a therapeutic composition that
includes a therapeutically effective amount of one or more agents
that reduce cellular invasion due to the interaction of OPN-5 kD
with CD44 (such as peptide fragments of OPN-5 kD or antibodies
specific for OPN-5 kD) further includes therapeutically effective
amounts of one or more other biologically active compounds.
Examples of biologically active compounds include, but are not
limited to: anti-neoplastic agents (such as chemotherapeutics),
antibiotics, alkylating agents, antioxidants, adjuvants, and so
forth (such as those listed below under "additional treatments").
However, one skilled in the art will appreciate that peptide
fragments of OPN-5 kD or antibodies specific for OPN-5 kD and the
other biologically active compounds can also be administered
separately (instead of in a single composition).
[0152] The therapeutically effective amount of the agents
administered can vary depending upon the desired effects and the
subject to be treated. In one example, the method includes daily
administration of at least 1 .mu.g of one or more peptide fragments
of OPN-5 kD to the subject (such as a human subject). In one
example, a human is administered at least 1 .mu.g of peptide daily
or at least 1 mg peptide daily of one or more OPN-5 kD fragments
(such as one or more of SEQ ID NOS: 5-8), such as 10 .mu.g to 100
.mu.g daily, 100 .mu.g to 1000 .mu.g daily, for example 10 .mu.g
daily, 100 .mu.g daily, or 1000 .mu.g daily. For example the
subject can receive at least 1 .mu.g (such as 1 .mu.g to 100 .mu.g,
5 .mu.g to 50 .mu.g, or 1 .mu.g to 1000 .mu.g) intravenously of
each of one or more OPN-5 kD fragments (such as one or more of SEQ
ID NOS: 5-8). In one example, the subject is administered at least
1 mg intramuscularly (for example in an extremity) or topically of
each of one or more OPN-5 kD fragments (such as one or more of SEQ
ID NOS: 5-8). For example, a human can be administered at least 1
.mu.g daily or at least 1 mg daily of one or more OPN-5 kD
fragments (such as one or more of SEQ ID NOS: 5-8), such as 10
.mu.g to 100 .mu.g daily, 100 .mu.g to 1000 .mu.g daily, for
example 10 .mu.g daily, 100 .mu.g daily, or 1000 .mu.g daily. In
one example, the subject is administered at least 1 .mu.g (such as
1 .mu.g to 100 .mu.g, 5 .mu.g to 50 .mu.g, or 1 .mu.g to 1000
.mu.g) intravenously of each of one or more OPN-5 kD fragments
(such as one or more of SEQ ID NOS: 5-8). In one example, the
subject is administered at least 1 mg intramuscularly (for example
in an extremity) or topically of each of one or more OPN-5 kD
fragments (such as one or more of SEQ ID NOS: 5-8). The dosage can
be administered in divided doses (such as 2, 3, or 4 divided doses
per day), or in a single dosage daily.
[0153] Therapeutic amounts of OPN-5 kD specific antibodies
disclosed herein (e.g., 5 kd106-13 D or a humanized form thereof, a
chimeric form thereof, or a fragment thereof) can also be
administered. In some examples, therapeutic amounts are amounts
which eliminate or reduce the patient's tumor burden, or which
prevent or reduce the proliferation of metastatic cells. The dosage
will depend on many parameters, including the nature of the tumor,
patient history, patient condition, the possible co-use of other
oncolytic agents, and methods of administration. Methods of
administration include injection (e.g., parenteral, subcutaneous,
intravenous, intraperitoneal, etc.) for which the antibodies are
provided in a nontoxic pharmaceutically acceptable carrier such as
water, saline, Ringer's solution, dextrose solution, 5% human serum
albumin, fixed oils, ethyl oleate, or liposomes. Typical dosages
may range from about 0.01 to about 20 mg/kg, such as from about 0.1
to about 10 mg/kg. Other methods of administration include oral and
transdermal (such as at least 1 mg, for example 1-1000 mg).
Acceptable carriers for oral ingestion include pharmaceutically
acceptable liquid carriers or pharmaceutically acceptable solid
carriers in the form of tablets, capsules, caplets, or gel-seals.
Other effective methods of administration and dosages may be
determined by routine experimentation and are within the scope of
this invention.
[0154] Therapeutic methods employing OPN-5 kD specific antibodies
or peptide fragments of OPN-5 kD can be combined with chemotherapy,
surgery, and radiation therapy, depending on type of the tumor,
patient condition, other health issues, and a variety of factors.
The methods can also include immunoconjugates for targeted
immunotoxin-mediated therapy, wherein OPN-5 kD antibodies are
covalently or non-covalently conjugated to various cytotoxic
agents, further enhancing toxicity to targeted cells. See, for
example, U.S. Pat. No. 5,872,223. Such agents, including various
bacterial toxins (e.g., Pseudomonas exotoxin), ricin A-chain,
daunorubicin, methotrexate, and ribosome inhibitors (e.g.,
trichosantin). Also, OPN-5 kD antibodies can be labeled with alpha,
beta, or Auger electron emitters, resulting in immunoconjugates for
targeted radiotherapy.
[0155] Thus, OPN-5 kD specific antibodies can be used in a variety
of methods and compositions for detecting and treating metastatic
disease.
[0156] In particular examples, the subject is administered the
therapeutic composition that includes OPN-5 kD specific antibodies,
or one or more peptide fragments of OPN-5 kD, on a multiple daily
dosing schedule, such as at least two consecutive days, 10
consecutive days, and so forth, for example for a period of weeks,
months, or years. In one example, the subject is administered the
therapeutic composition for a period of at least 30 days, such as
at least 2 months, at least 4 months, at least 6 months, at least
12 months, at least 24 months, or at least 36 months.
Additional Treatments
[0157] In particular examples, prior to, during, or following
administration of a therapeutic amount of an agent that reduces
cellular invasion due to the interaction of OPN-5 kD with CD44
(such as one or more peptide fragments of OPN-5 kD or OPN-5 kD
antibodies), the subject can receive one or more other therapies.
In one example, the subject receives one or more treatments to
remove or reduce the tumor prior to administration of a therapeutic
amount of one or more agents that reduce cellular invasion due to
the interaction of OPN-5 kD with CD44.
[0158] Examples of such therapies include, but are not limited to,
surgical treatment for removal or reduction of the tumor (such as
surgical resection, cryotherapy, or chemoembolization), as well as
anti-tumor pharmaceutical treatments which can include
radiotherapeutic agents, anti-neoplastic chemotherapeutic agents,
antibiotics, alkylating agents and antioxidants, kinase inhibitors,
and other agents. In one example, at least part of the tumor is
surgically or otherwise excised or reduced in size or volume prior
to administering the therapeutically effective amount of the
antibody or peptide. Particular examples of additional therapeutic
agents can that can be used include microtubule binding agents, DNA
intercalators or cross-linkers, DNA synthesis inhibitors, DNA
and/or RNA transcription inhibitors, antibodies, enzymes, enzyme
inhibitors, gene regulators, and angiogenesis inhibitors. These
agents (which are administered at a therapeutically effective
amount) and treatments can be used alone or in combination. Methods
and therapeutic dosages of such agents are known to those skilled
in the art, and can be determined by a skilled clinician.
[0159] "Microtubule binding agent" refers to an agent that
interacts with tubulin to stabilize or destabilize microtubule
formation thereby inhibiting cell division. Examples of microtubule
binding agents that can be used in conjunction with the disclosed
therapy include, without limitation, paclitaxel, docetaxel,
vinblastine, vindesine, vinorelbine (navelbine), the epothilones,
colchicine, dolastatin 15, nocodazole, podophyllotoxin and
rhizoxin. Analogs and derivatives of such compounds also can be
used and are known to those of ordinary skill in the art. For
example, suitable epothilones and epothilone analogs are described
in International Publication No. WO 2004/018478. Taxoids, such as
paclitaxel and docetaxel, as well as the analogs of paclitaxel
taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be
used.
[0160] Suitable DNA and/or RNA transcription regulators, including,
without limitation, actinomycin D, daunorubicin, doxorubicin and
derivatives and analogs thereof also are suitable for use in
combination with the disclosed therapies.
[0161] DNA intercalators and cross-linking agents that can be
administered to a subject include, without limitation, cisplatin,
carboplatin, oxaliplatin, mitomycins, such as mitomycin C,
bleomycin, chlorambucil, cyclophosphamide and derivatives and
analogs thereof.
[0162] DNA synthesis inhibitors suitable for use as therapeutic
agents include, without limitation, methotrexate,
5-fluoro-5'-deoxyuridine, 5-fluorouracil and analogs thereof.
[0163] Examples of suitable enzyme inhibitors include, without
limitation, camptothecin, etoposide, formestane, trichostatin and
derivatives and analogs thereof.
[0164] Suitable compounds that affect gene regulation include
agents that result in increased or decreased expression of one or
more genes, such as raloxifene, 5-azacytidine,
5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone
and derivatives and analogs thereof.
[0165] "Angiogenesis inhibitors" include molecules, such as
proteins, enzymes, polysaccharides, oligonucleotides, DNA, RNA, and
recombinant vectors, and small molecules that function to reduce or
even inhibit blood vessel growth. Angiogenesis is implicated in
most types of human solid tumors. Angiogenesis inhibitors are known
in the art and examples of suitable angiogenesis inhibitors
include, without limitation, angiostatin K1-3, staurosporine,
genistein, fumagillin, medroxyprogesterone, suramin,
interferon-alpha, metalloproteinase inhibitors, platelet factor 4,
somatostatin, thromobospondin, endostatin, thalidomide, and
derivatives and analogs thereof.
[0166] Exemplary kinase inhibitors include Gleevac, Iressa, and
Tarceva that prevent phosphorylation and activation of growth
factors.
[0167] Antibodies that can be used include Herceptin and Avastin
that block growth factors and the angiogenic pathway.
[0168] Other therapeutic agents, for example anti-tumor agents,
that may or may not fall under one or more of the classifications
above, also are suitable for administration in combination with the
disclosed therapies (such as those that include one or more peptide
fragments of OPN-5 kD or an OPN-5 kD specific antibody). By way of
example, such agents include adriamycin, apigenin, rapamycin,
zebularine, cimetidine, and derivatives and analogs thereof.
[0169] In one example, the therapeutic peptide composition (such as
OPN-5 kD or peptide fragments thereof) or is injected into the
subject in the presence of an adjuvant, thus generating an immune
response to OPN-5 kD wherein such immune response decreases OPN-5
kD binding to CD44. An adjuvant is an agent that when used in
combination with an immunogenic agent augments or otherwise alters
or modifies a resultant immune response. In some examples, an
adjuvant increases the titer of antibodies induced in a subject by
the immunogenic agent. In one example, the one or more peptides are
administered to the subject as an emulsion with IFA and sterile
water for injection (for example an intravenous or intramuscular
injection). Incomplete Freund's Adjuvant (Seppic, Inc.) can be used
as the Freund's Incomplete Adjuvant (IFA) (Fairfield, N.J.). In
some examples, IFA is provided in 3 ml of a mineral oil solution
based on mannide oleate (Montanide ISA-51). At the time of
injection, the peptide(s) is mixed with the Montanide ISA.51 and
then administered to the subject. Other adjuvants can be used, for
example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide,
saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein,
hemocyanin, a mycobacterial antigen, and combinations thereof.
[0170] In some examples, the subject receiving a therapeutic
composition (such as one or more peptide fragments of OPN-5 kD or
OPN-5 kD antibodies) is also administered interleukin-2 (IL-2), for
example via intravenous administration. In particular examples,
IL-2 (Chiron Corp., Emeryville, Calif.) is administered at a dose
of at least 500,000 IU/kg as an intravenous bolus over a 15 minute
period every eight hours beginning on the day after administration
of the peptides and continuing for up to 5 days. Doses can be
skipped depending on subject tolerance.
[0171] In some examples, the therapeutic compositions can be
co-administered with a fully human antibody to cytotoxic
T-lymphocyte antigen-4 (anti-CTLA-4). In some examples subjects
receive at least 1 mg/kg anti-CTLA-4 (such as 3 mg/kg every 3 weeks
or 3 mg/kg as the initial dose with subsequent doses reduced to 1
mg/kg every 3 weeks).
[0172] In one example, at least a portion of the tumor (such as a
metastatic HCC) is surgically removed (for example via
cryotherapy), irradiated, chemically treated (for example via
chemoembolization) or combinations thereof, prior to administration
of the disclosed therapies. For example, a subject having HCC can
have all or part of the tumor surgically excised prior to
administration of the disclosed therapies. In another particular
example, the subject has HCC and is administered radiation therapy,
chemoembolization therapy, or both, prior to administration of the
disclosed therapies.
Therapeutic Compositions
[0173] Another aspect of the disclosure includes pharmaceutical
compositions prepared for administration to a subject and which
include a therapeutically effective amount of one or more of the
currently disclosed compounds. Such compositions can also be part
of a kit. For example, compositions that include a therapeutic
amount of an agent that decreases or inhibits cellular invasion
that results from the interaction of OPN-5 kD with CD44 receptor
can be formulated for use in treating a tumor that overexpresses
OPN, such as HCC. Particular examples of such agents include OPN-5
kD specific antibodies and peptide fragments of OPN-5 kD, such as
the peptide sequences shown in SEQ ID NOS: 5-8.
[0174] In one example, compositions include a therapeutic amount of
one or more agents that decrease or inhibit tumor cellular invasion
(for example a reduction of at least 10%, at least 20%, at least
50%, or even at least 90%) such as by decreasing the binding of
OPN-5 kD to the CD44 receptor. Such compositions can include one or
more additional biologically active agents, one or more
biologically inactive compounds, or combinations thereof. Examples
of such biologically active agents are described above, and can
include anti-neoplastic agents. Examples of biologically inactive
agents are described above, and can include pharmaceutically
acceptable carriers. In a specific example, the therapeutic
composition includes an antibody to cytotoxic T-lymphocyte
antigen-4 (anti-CTLA-4).
[0175] Particular examples of agents that can be used in the
disclosed compositions include peptide fragments of OPN-5 kD that
decrease tumor cellular invasion resulting from the interaction of
OPN-5 kD with CD44 receptor. One example of OPN-5 kD peptide
fragments includes peptides of 5 to 60 amino acids that have at
least 5 contiguous amino acids of OPN-5 kD (such as at least 5
contiguous amino acids of SEQ ID NO: 4), such as at least 6, at
least 7, at least 8, at least 9, at least 10, at least 12, at least
15, or even at least 20 contiguous amino acids of OPN-5 kD, for
example 5-30, 5-25, 6-20, 6-12, or 8-10 contiguous amino acids of
OPN-5 kD (such as this number of contiguous amino acids of SEQ ID
NO: 4). Exemplary examples of such peptides are shown in SEQ ID
NOS: 5-8.
[0176] In a particular example, the composition includes at least
two peptide fragments of OPN-5 kD. For example, the composition can
include a mixture of at least 2 of the peptides shown in SEQ ID
NOS: 5-8, such as a composition that includes 2, 3, or 4 of these
peptides. Exemplary combinations include the peptides shown in SEQ
ID NOS: 5 and 6, SEQ ID NOS: 5 and 7, SEQ ID NOS: 5 and 8, SEQ ID
NOS: 6 and 7, SEQ ID NOS: 6 and 8, SEQ ID NOS: 7 and 8, SEQ ID NOS:
5, 6, and 7, SEQ ID NOS: 5, 6, and 8, SEQ ID NOS: 5, 7, and 8, SEQ
ID NOS: 6, 7, and 8, or SEQ ID NOS: 5, 6, 7, and 8. In one example,
the composition includes one or more peptides consisting of the
amino acid sequence shown in SEQ ID NO: 5, 6, 7, or 8.
[0177] In particular examples, the composition includes 1-1000
.mu.g of one or more peptide fragments of OPN-5 kD that decrease
tumor cellular invasion resulting from the interaction of OPN-5 kD
with CD44 receptor, such as 1-1000 .mu.g of one or more of SEQ ID
NOS: 5-8. In some examples, the composition includes 1-1000 mg of
one or more peptide fragments of OPN-5 kD that decrease tumor
cellular invasion resulting from the interaction of OPN-5 kD with
CD44 receptor, such as 1-1000 mg of one or more of SEQ ID NOS:
5-8.
[0178] In another particular example, the composition includes one
or more OPN-5 kD specific antibodies, such as 5 kd106-13 D (or
other monoclonal antibody or humanized form thereof that recognizes
the epitope shown in amino acids 30-36 of SEQ ID NO: 4). Such
antibodies bind to OPN-5 kD or fragments thereof and inhibit or
decrease binding to CD44 receptors. In particular examples, the
composition includes 1-1000 .mu.g or 1-1000 mg of one or more OPN-5
kD specific antibodies that decrease tumor cellular invasion
resulting from the interaction of OPN-5 kD with CD44 receptor.
Details on such antibodies are provided below.
Methods of Diagnosing and Prognosing a Tumor
[0179] Metastasis is a major complication in the pathogenesis of
tumors, such as HCC, and is typically indicative of poor prognosis.
It is shown herein that OPN-5 kD abundance in HCC cell lines
correlates with degree of metastatic potential. Without wishing to
be bound to a particular theory, it is proposed that the
interaction of OPN and MMP-9 is related to enhanced HCC tumor cell
metastasis. The abundance of the OPN-5 kD fragment in HCC cells
correlates to degree of metastatic potential. It is also shown
herein that OPN-c mRNA expression correlates with metastatic HCC
but not non-metastatic HCC in non-cancerous tissue adjacent to
primary HCC lesions. Based on these observations, methods of
diagnosing or prognosing a tumor that overexpresses OPN, MMP-9, or
both, based on detecting OPN-5 kD, OPN-c, or both, are disclosed.
In some examples, such methods can be used to identify those
subjects that will benefit from the disclosed treatment methods.
For example, such diagnostic methods can be performed prior to the
subject undergoing the treatments described herein.
[0180] In some examples the method is performed in vitro, for
example when the biological sample is removed from the subject. In
some examples the method is carried out in vivo for example by
administering the disclosed antibodies, humanized form thereof or
fragment thereof to a subject, such as a subject that has or is
suspected of having an OPN-expressing tumor, such as HCC.
[0181] In one example, detection of the OPN-5 kD fragment (such as
SEQ ID NO: 4 or a variant thereof, such as a polymorphism thereof)
in a biological sample from the subject is used to diagnose or
prognose a tumor that overexpresses OPN, MMP-9, or both (such as
HCC). Methods of detecting a peptide in a sample, such as OPN-5 kD,
are known in the art and are routine. In some examples, the
relative amount of OPN-5 kD present is determined, for example by
quantitating the amount of OPN-5 kD present. For example, the
relative or absolute quantity of OPN-5 kD in a sample can be
determined.
[0182] In another example, detection of OPN-c mRNA (such as an mRNA
corresponding to SEQ ID NO: 3 or 9, or a variant thereof, such as a
polymorphism thereof) in a biological sample from the subject is
used to diagnose or prognose a tumor that overexpresses OPN, MMP-9,
or both (such as metastatic HCC). Methods of detecting a nucleic
acid molecule in a sample, such as OPN-c, are known in the art and
are routine. In some examples, the relative amount of OPN-c mRNA
present is determined, for example by quantitating the amount of
OPN-c mRNA present. For example, the relative or absolute quantity
of OPN-c mRNA in a sample can be determined. However, one skilled
in the art will recognize that OPN-c protein can be detected as an
alternative to detecting OPN-c nucleic acid molecules. In some
examples, OPN-c expression is measured in a tumor sample and an
adjacent non-tumor sample, wherein an increase in OPN-c expression
(such as an increase of at least 2-fold, at least 3-fold, or at
least 5-fold) in the tumor sample relative to OPN-c expression in
the non-tumor sample indicates that the subject has a tumor with
increased metastatic potential. In other examples, OPN-c and OPN-a
expression are measured in the tumor sample, wherein an increase in
OPN-c expression (such as an increase of at least 2-fold, at least
3-fold, or at least 5-fold) relative to OPN-a expression in the
tumor sample indicates that the subject has an OPN-overexpressing
tumor.
[0183] In some examples, detection of both OPN-5 kD protein and
OPN-c mRNA (an in some examples also OPN-a mRNA) in one or more
biological samples obtained from the subject (such as serum or a
tumor sample or adjacent non-tumor sample) are used to diagnose or
prognose a tumor that overexpresses OPN, MMP-9, or both (such as
metastatic HCC).
Biological Samples
[0184] A biological sample is typically obtained from a mammal,
such as a rat, mouse, cow, dog, guinea pig, rabbit, or primate,
such as a human. Thus is some examples a subject, such as a human
subject, is selected and a biological sample from that subject is
tested for the presence of an OPN-expressing tumor (for example by
detecting OPN-5 kD, OPN-c, and/or OPN-a). Methods of obtaining a
biological sample from a subject are known in the art. For example,
methods of obtaining a blood sample or a fraction thereof (such as
serum) are routine. Similarly, a sample from a tumor (or adjacent
non-tumor tissue) that contains cellular material can be obtained
by surgical excision of all or part of the tumor, by collecting a
fine needle aspirate from the tumor, as well as other methods known
in the art. If desired, the sample can be concentrated or purified
before use. For example, proteins or nucleic acids can be isolated
from the sample. In some examples, the sample is filtered before
use, for example to remove undesired proteins (e.g., fractionating
serum to remove full-length OPN while retaining OPN-5 kD).
Alternatively, the sample can be used directly. In particular
examples, a serum sample obtained from the subject is analyzed to
determine if it contains detectable levels of OPN-5 kD. In
particular examples, a tumor sample obtained from the subject is
analyzed to determine if it contains detectable levels of OPN-c
(and in some examples also OPN-a) mRNA or protein.
Detection of OPN-5 kD Peptide In Vitro
[0185] In particular examples, a sample obtained from the subject
is analyzed to determine if it contains detectable levels of the
OPN-5 kD fragment, such as a serum sample.
[0186] Methods of detecting proteins are routine. In some examples,
immunoassays are used to detect the presence of the OPN-5 kD
protein fragment in the sample. Generally, immunoassays include the
use of one or more specific binding agents (such as antibodies)
that can substantially only bind to OPN-5 kD. Such binding agents
can include a detectable label (such as a radiolabel, fluorophore
or enzyme), that permits detection of the binding to the protein.
Exemplary immunoassays that can be used include, but are not
limited to: Western blotting, ELISA, fluorescence microscopy, and
flow cytometry.
[0187] In one example, the specific binding agent is an antibody,
such as a polyclonal or monoclonal antibody, or fragment thereof.
In some examples, the antibody is a humanized antibody. In some
examples, the antibody is a chimeric antibody. If desired, the
antibody can include a detectable label to permit detection and in
some cases quantitation of the OPN-5 kD/antibody complex.
[0188] In a particular example, the antibody used is 5 kd106-13 D.
For example, the method can include contacting the biological
sample (e.g., serum) with the monoclonal 5 kd106-13 D antibody, a
chimeric form thereof, a humanized form thereof, or a functional
fragment thereof under conditions in which an immune complex will
form between the 5 kd106-13 D antibody, chimeric form thereof,
humanized form thereof, or functional fragment thereof and the
OPN-5 kD fragment. In some examples, the presence (or absence) of
the immune complex is then detected. The presence of the immune
complex indicates the presence of the OPN-5 kD fragment, and thus
an OPN-expressing or -overexpressing tumor such as HCC. In some
embodiments a second antibody (such as an antibody that recognizes
a mouse IgG) (which can be detectably labeled) that specifically
binds the 5 kd106-13 D antibody, a chimeric form thereof, or a
humanized form thereof or a functional fragment thereof is used to
detect OPN-5 kD.
[0189] The presence of detectable signal above background or
control levels indicates that the presence of OPN-5 kD peptide in
the sample. For example, the level of OPN-5 kD detected can be
compared to a control or reference value, such as a value that
represents a level of OPN-5 kD expected if a tumor that
overexpresses OPN is present in the subject (such as metastatic
HCC), a level of OPN-5 kD expected if a tumor that overexpresses
OPN is absent in the subject (such as HCC or a normal subject), a
level of OPN-5 kD expected if a tumor that overexpresses OPN is has
metastasized in the subject (such as metastatic HCC), or
combinations thereof.
[0190] In some examples, full-length OPN is also measured in the
sample, and the expression levels of OPN and OPN-5 kD compared, for
example to ratios expected for healthy individuals, subjects with a
metastatic OPN-overexpressing tumor, or subjects with an
non-metastic OPN-expressing tumor, wherein the tested ratio is
compared to the control ratio, and the control ratio that is most
similar to the test ratio indicates the diagnosis of the
subject.
[0191] In some examples, detection of OPN-5 kD at a level of at
least twice (such as at least three or at least five times) that
observed in a subject not having a tumor, indicates that that
subject has a tumor that overexpresses OPN, has a tumor with
metastatic potential, has a tumor that has metastasized, has a poor
prognosis, or combinations thereof. For example, if serum levels of
OPN-5 kD from a subject are twice that observed for a subject not
having a tumor, such as an increase of at least 100%, at least
200%, or at least 500%, this indicates that the subject has a tumor
that overexpresses OPN, has a tumor with metastatic potential, has
a tumor that has metastasized, has a poor prognosis, or
combinations thereof.
[0192] For example, if the level of OPN-5 kD detected in the
subject's sample is significantly less than the level of OPN-5 kD
expected if a tumor that overexpresses OPN is present in the
subject, is similar to or less than the level of OPN-5 kD expected
if a tumor that overexpresses OPN is not present in the subject, or
both, this indicates that the subject does not have a tumor that
overexpresses OPN. In another example, if the level of OPN-5 kD
detected in the subject's sample is similar or greater than the
level of OPN-5 kD expected if a tumor that overexpresses OPN is
present in the subject, is significantly greater than the level of
OPN-5 kD expected if a tumor that overexpresses OPN is not present
in the subject, or both, this indicates that the subject has a
tumor that overexpresses OPN, such as HCC. In another example, if
the level of OPN-5 kD detected in the subject's sample is similar
or greater than the level of OPN-5 kD expected if a tumor that
overexpresses OPN is present in the subject, is significantly
greater than the level of OPN-5 kD expected if a tumor that
overexpresses OPN is not present in the subject, is similar to or
greater than the level of a level of OPN-5 kD expected if a tumor
that overexpresses OPN is has metastasized in the subject, or
combinations thereof, this indicates that the subject has a
metastatic tumor that overexpresses OPN, such as metastatic
HCC.
Detection of OPN-5 kD In Vivo
[0193] OPN-5 kD specific antibodies can also be used in vivo for
diagnosis. For example, the 5 kd106-13 D antibody, a chimeric form
thereof, or a humanized form thereof or a fragment thereof
disclosed herein can also be used to detect OPN-expressing or
over-expressing tumors (e.g., HCC), in vivo. The antibodies
disclosed herein can also be used to detect HCC and metastatic HCC
in vivo. In one embodiment, a 5 kd106-13 D antibody, a chimeric
form thereof, or a humanized form thereof or a fragment thereof is
administered to the subject for a sufficient amount of time for the
antibody to localize to the tumor in the subject and to form an
immune complex with the tumor. The immune complex can then be
detected for example radiolocalization, radioimaging, MRI, PET
scan, or fluorescence imaging, for example by using a detectibly
labeled antibody, humanized form thereof or functional fragment
thereof. Once detected, in an ectopic location (as in a tumor) the
test results can be used to assist in or guide surgical or other
excision of a tumor.
[0194] In vivo imaging methods can also be utilized with the OPN-5
kD antibodies disclosed herein. These technologies include magnetic
resonance imaging (for example using a biotinylated antibody and
avidin-iron oxide), positron emission tomography (for example using
an .sup.111indium-labeled monoclonal antibody), and optical imaging
(for example using luciferase or green fluorescent protein labeled
antibodies). In one example, magnetic resonance imaging is
utilized. In the setting of magnetic resonance imaging, contrast
agent detection can be greatly impacted by magnetic resonance
scanner field strength. Increased field strengths provide
improvements by orders of magnitude in the ability to detect
contrast agents (Hu et al., Annu Rev Biomed Eng. 6:157-184, 2004;
Wedeking et al., Magn. Reson. Imaging. 17:569-575, 1999). For
example, the limit of detection of gadolinium at 2 tesla (T) is
.about.30 .mu.M. At 4T the limit of detection is reduced to
.about.1 .mu.M. With newly available 7 to 12T scanners one would
expect to detect low (10-100) nM concentrations of this contrast
agent. Similar sensitivity can also be identified using contrast
agents such as iron oxide.
Detection of OPN-c and OPN-a
[0195] In particular examples, a sample obtained from the subject
is analyzed to determine if it contains detectable levels of OPN-c
and in some examples also OPN-a, such as a tumor sample, a sample
adjacent to the tumor, or both. In particular examples, OPN-a and
OPN-c nucleic acid molecules (such as mRNA) are measured. However,
one skilled in the art will appreciate that OPN-c protein could
also be measured, for example using routine immunoassays known in
the art (such as those described above).
[0196] Methods of detecting nucleic acid molecules are routine. In
particular examples, a tumor sample and a sample adjacent to the
tumor obtained from the subject are analyzed to determine if each
contains detectable levels of OPN-c nucleic acid molecules, such as
cDNA or mRNA. For example, assays that permit detection of nucleic
acids can be used. Exemplary assays that can be used include, but
are not limited to: Northern blotting, Southern blotting, PCR (such
as RT-PCR), and DNA arrays. For example, OPN-c can be amplified
from a sample using PCR, and the amplicons detected and in some
examples quantitated, wherein the presence of detectable amplicons
above background or control levels indicates that the tumor (or
adjacent tissue) expresses OPN-c nucleic acid molecules. In one
example, a nucleic acid probe that hybridizes to an OPN-c nucleic
acid is contacted with the sample. For example, the probe can be
incubated with the sample under high stringency conditions (such as
when the hybridization is performed at about 42.degree. C. in a
hybridization solution containing 25 mM KPO.sub.4 (pH 7.4),
5.times.SSC, 5.times.Denhart's solution, 50 .mu.g/mL denatured,
sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and
1-15 ng/mL probe (about 5.times.10.sup.7 cpm/.mu.m), while the
washes are performed at about 65.degree. C. with a wash solution
containing 0.2.times.SSC and 0.1% sodium dodecyl sulfate), wherein
the presence of detectable signal from the probe above background
or control levels indicates that the tumor (or adjacent tissue)
expresses OPN-c nucleic acid molecules.
[0197] The presence of detectable signal above background or
control levels indicates that the presence of OPN-c nucleic acid
molecules in the sample. For example, the level of OPN-c detected
can be compared to a control or reference value, such as a value
that represents a level of OPN-c expected if a tumor is or is not
metastatic (such as metastatic HCC). In one example, the level of
OPN-c detected in a tumor sample is compared to the level of OPN-c
detected in an adjacent non-tumor sample.
[0198] In some examples, detection of at least twice (such as at
least 3-times or at least 5-times) the relative amount of OPN-c
mRNA observed in a tumor sample, as compared to the relative amount
of OPN-c mRNA in an adjacent non-tumor sample from the same
subject, indicates that that subject has a tumor with metastatic
potential, has a tumor that has metastasized, has a poor prognosis,
or combinations thereof. For example, if tumor levels of OPN-c mRNA
are twice that observed for an adjacent non-tumor sample, such as
an increase of at least 100%, at least 200%, or at least 500%, this
indicates that the subject has a tumor with metastatic potential,
has a tumor that has metastasized, has a poor prognosis, or
combinations thereof.
[0199] In some examples, detection of statistically similar
relative amounts of OPN-c mRNA observed in a tumor sample, as
compared to the relative amount of OPN-c mRNA in an adjacent
non-tumor sample from the same subject, indicates that that subject
does not have a tumor with metastatic potential, does not have a
tumor that has metastasized, has a good prognosis, or combinations
thereof.
[0200] In some examples, the ratio of OPN-a to OPN-c in tumor cells
is determined. It is shown herein that the ratio for OPN-a versus
OPN-c in metastatic tumors is slightly lower than for
non-metastatic tumors, indicating higher levels of OPN-c in
metastasis (for example a ratio of 2.27 versus 2.85). Thus methods
are provided for measuring OPN-a and OPN-c expression in a tumor
sample. OPN-a can be detected using methods similar to that
described above for OPN-c. Expression levels of OPN-c expression
and OPN-a expression in the tumor sample are compared, wherein a
statistically significant increase in OPN-c expression (such as an
increase of at least 20%, at least 50%, or at least 80%) in the
tumor sample compared to the amount of OPN-a expression in the
tumor sample indicates that the subject has an OPN-expressing
tumor.
Monitoring Response to Therapy
[0201] The disclosed OPN-5 kD antibodies and fragments thereof can
be used to monitor response to therapy. The number and or mass of
OPN-expressing tumor cells (e.g., HCC cells), such as the cells
present in a subject, can be determined using the methods disclosed
herein. In one embodiment, an increase in the number or mass of
OPN-expressing tumor cells (e.g., HCC cells), as compared to a
control, such as the number or mass of OPN-expressing tumor cells
(e.g., HCC cells) at an earlier time point, indicates that the
tumor is progressing and that the therapy as not effective in
reducing tumor burden. Conversely, a decrease in the number or mass
of OPN-expressing tumor cells (e.g., HCC cells), as compared to a
control, such as the number or mass of OPN-expressing tumor cells
(e.g., HCC cells) at an earlier time point, indicates that the
tumor is regressing and that the therapy is effective. A control
can be a standard value, or the number or mass of OPN-expressing
tumor cells (e.g., HCC cells) in a sample from a subject not
afflicted with a tumor or the number or mass of OPN-expressing
tumor cells (e.g., HCC cells) in a sample from the subject at an
earlier time point, for example prior to therapy.
Kits
[0202] Provided by this disclosure are kits that can be used to
diagnose, prognose, or treat a tumor that overexpresses OPN (or
combinations thereof). The disclosed kits can include instructional
materials disclosing means of use of the compositions in the kit.
The instructional materials can be written, in an electronic form
(such as a computer diskette or compact disk) or can be visual
(such as video files).
[0203] Kits are provided that can be used in the therapies and
diagnostic assays disclosed herein. For example, kits can include
one or more of the disclosed therapeutic compositions (such as a
composition including one or more peptide fragments of OPN-5 kD or
OPN-5 kD specific antibodies), one or more of the disclosed
diagnostic compositions (such as an antibody that specifically
binds OPN-5 kD), or combinations thereof. Such agents can be
present in separate vials.
[0204] One skilled in the art will appreciate that the kits can
include other agents to facilitate the particular application for
which the kit is designed. Thus, for example, the kit may
additionally contain means of detecting a label (such as enzyme
substrates for enzymatic labels, filter sets to detect fluorescent
labels, appropriate secondary labels such as a secondary antibody,
or the like). The kits may additionally include buffers and other
reagents routinely used for the practice of a particular method.
Such kits and appropriate contents are well known to those of skill
in the art.
[0205] In one example, a kit is provided that includes reagents for
detecting OPN-5 kD in a biological sample, such as serum. Kits for
detecting OPN-5 kD can include one or more antibodies that
specifically bind OPN-5 kD, such as any of the antibodies disclosed
herein. For example, an OPN-5 kD-specific antibody disclosed herein
(e.g., 5 kd106-13 D), a chimeric form thereof, a humanized form
thereof, or a fragment thereof can be part of the disclosed kits.
In some embodiments, an OPN-5 kD antibody fragment, such as an Fv
fragment is included in the kit. In one example, such as for in
vivo uses, the antibody can be a scFv fragment. In a further
embodiment, the antibody is labeled (for example, with a
fluorescent, radioactive, or an enzymatic label). Such a diagnostic
kit can additionally contain means of detecting a label (such as
enzyme substrates for enzymatic labels, filter sets to detect
fluorescent labels, appropriate secondary labels such as a
secondary antibody, or the like), as well as buffers and other
reagents routinely used for the practice of a particular diagnostic
method.
[0206] In one example, the diagnostic kit comprises an immunoassay.
Although the details of the immunoassays may vary with the
particular format employed, the method of detecting the OPN-5 kD
fragment in a biological sample generally includes the steps of
contacting the sample with an antibody which specifically reacts
with OPN-5 kD (e.g., 5 kd106-13 D) under immunologically reactive
conditions. The antibody is allowed to specifically bind under
immunologically reactive conditions to form an immune complex, and
the presence of the immune complex (bound antibody) is detected
directly or indirectly.
[0207] In one example, a kit is provided for treating a tumor that
overexpresses OPN, such as HCC. For example, such kits can include
one or more of the disclosed therapeutic compositions (such as a
composition including an OPN-5 kD specific antibody or including
one or more peptide fragments of OPN-5 kD). Such a diagnostic kit
can additionally contain other therapeutic molecules, such as
therapeutic doses of IL-2, anti-CTLA-4, an adjuvant, or
combinations thereof.
OPN-5 kD Antibodies
[0208] The present disclosure provides antibodies that specifically
bind OPN-5 kD, such as bind to the epitope EELNGAY (amino acids
30-36 of SEQ ID NO: 4). One exemplary anti-OPN-5 kD monoclonal
antibody is 5 kD106-13 D. The disclosed OPN-5 kd antibodies are
useful for both therapeutic and diagnostic purposes. In some
examples OPN-5 kD specific antibodies have an equilibrium constant
(K.sub.d) of 1 nM or less. For example, antibodies are provided
that bind human OPN-5 kD with a binding affinity of
0.1.times.10.sup.-8 M, at least about 0.3.times.10.sup.-8M, at
least about 0.5.times.10.sup.-8M, at least about
0.75.times.10.sup.-8 M, at least about 1.0.times.10.sup.-8M, at
least about 1.3.times.10.sup.-8 Mat least about
1.5.times.10.sup.-8M, or at least about 2.0.times.10.sup.-8 M. In
additional examples, the antibody (such as a human monoclonal)
binds an epitope of OPN-5 kD with an equilibrium disassociation
constant (K.sub.d) of 1 nM or less. In particular examples, the
antibody can be used to diagnose a tumor, or be used to predict the
metastatic potential of the tumor. In other examples, the
antibodies can be used in therapeutic compositions to treat a
subject. The disclosed antibodies can be part of a kit, such as a
kit used to diagnose a tumor, such as HCC (such as metastatic
HCC).
[0209] In one example, the antibody is a monoclonal antibody. The
monoclonal antibody can be of any isotype, such as an IgM or an IgG
antibody, for example IgG.sub.1 or an IgG.sub.2. The class of an
antibody that specifically binds OPN-5 kD can be switched with
another. In one aspect, a nucleic acid molecule encoding V.sub.L or
V.sub.H is isolated using methods well-known in the art, such that
it does not include any nucleic acid sequences encoding the
constant region of the light or heavy chain, respectively. The
nucleic acid molecule encoding V.sub.L or V.sub.H is then
operatively linked to a nucleic acid sequence encoding a C.sub.L or
C.sub.H from a different class of immunoglobulin molecule. This can
be achieved using a vector or nucleic acid molecule that includes a
C.sub.L or C.sub.H chain, as known in the art. For example, an
antibody that specifically binds OPN-5 kD that was originally IgM
can be class switched to an IgG. Class switching can be used to
convert one IgG subclass to another, such as from IgG.sub.1 to
IgG.sub.2.
[0210] Isolated monoclonal antibodies specific for the OPN-5 kD
fragment are provided that are produced by the hybridoma 5 kd106-13
D, as are humanized forms of the 5 kd106-13 D antibody, chimeras of
the 5 kd106-13 D antibody, and functional fragments of 5 kd106-13
D. The 5 kd106-13 D antibody specifically binds the OPN-5 kD
fragment produced by HCC cells. Generally, the monoclonal
antibodies produced by the 5 kd106-13 D hybridoma include a
variable heavy (V.sub.H) and a variable light (V.sub.L) chain and
specifically bind the OPN-5 kD fragment. For example, the 5
kd106-13 D antibody can specifically bind the OPN-5 kD fragment,
such as an epitope thereof (e.g., amino acids 30-36 of SEQ ID NO:
4) with an affinity constant of at least 10.sup.6 M.sup.-1, such as
at least 10.sup.7 M.sup.-1, at least 10.sup.8 M.sup.-1, at least
5.times.10.sup.8 M.sup.-1, or at least 10.sup.9 M.sup.-1. Hybridoma
cells and their progeny that secrete the monoclonal antibody 5
kd106-13 D are also encompassed by this disclosure.
[0211] Antibody fragments are encompassed by the present
disclosure, such as Fab, F(ab').sub.2, and Fv which include a heavy
chain and light chain variable region and are capable of binding
the epitopic determinant on OPN-5 kD (e.g., EELNGAY, amino acids
30-36 of SEQ ID NO: 4). These antibody fragments retain the ability
to selectively bind with the antigen. Methods of making these
fragments are known in the art (see for example, Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York, 1988). In a specific example, the antibody is an Fv antibody.
In other specific examples, the functional fragment of the
monoclonal antibody (e.g., 5 kd106-13 D) is a scFV fragment, an
scFV.sub.2 fragment, an Fv fragment, an Fab fragment, or an
F(ab').sub.2 fragment.
[0212] The production of chimeric antibodies, which include a
framework region from one antibody and the CDRs or SDRs from a
different antibody, is well known in the art. Thus chimeric and
humanized forms of anti-OPN-5 kD antibodies (e.g., the 5 kd106-13 D
antibody) are provided herein. These antibodies include the CDRs
(or SDRs) of an antibody specific for an epitope of the OPN-5 kD
fragment (e.g., the 5 kd106-13 D antibody) and framework regions
from a different antibody. In one example, the framework regions
are human. In some embodiments, a humanized antibody that
specifically binds the OPN-5 kD fragment is a humanized form of the
5 kd106-13 D monoclonal antibody (or other antibody specific for an
epitope of the OPN-5 kD fragment) or a functional fragment thereof.
In one example the sequence of the specificity determining regions
(SDRs) of each CDR from the 5 kd106-13 D monoclonal antibody (or
other antibody specific for an epitope of the OPN-5 kD fragment) is
determined. Residues outside the SDRs (non-ligand contacting sites)
can be substituted and the monoclonal antibody retains its ability
to bind the OPN-5 kD fragment. The antibody or antibody fragment
can be a humanized immunoglobulin having complementarity
determining regions (CDRs) from the 5 kd106-13 D monoclonal
antibody (or other antibody specific for an epitope of the OPN-5 kD
fragment) produced by the above-described hybridoma and
immunoglobulin and heavy and light chain variable region frameworks
from human acceptor immunoglobulin heavy and light chain
frameworks. Generally, the humanized immunoglobulin specifically
binds to the OPN-5 kD fragment or an epitope thereof (such as
EELNGAY, amino acids 30-36 of SEQ ID NO: 4) with an affinity
constant of at least 10.sup.7 M.sup.-1, such as at least 10.sup.8
M.sup.-1 at least 5.times.10.sup.8 M.sup.-1 or at least 10.sup.9
M.sup.-1.
[0213] Humanized monoclonal antibodies can be produced by
transferring donor CDRs from heavy and light variable chains of the
donor mouse immunoglobulin (such as the 5 kd106-13 D monoclonal
antibody) into a human variable domain, and then substituting human
residues in the framework regions when required to retain affinity.
The use of antibody components derived from humanized monoclonal
antibodies obviates potential problems associated with the
immunogenicity of the constant regions of the donor antibody.
Techniques for producing humanized monoclonal antibodies are
described, for example, by Jones et al., Nature 321:522, 1986;
Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science
239:1534, 1988; Carter et al., Proc. Nat'l Acad. Sci. U.S.A.
89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singer
et al., J. Immunol. 150:2844, 1993. The antibody may be of any
isotype, but in several embodiments the antibody is an IgG,
including but not limited to, IgG.sub.1, IgG.sub.2, IgG.sub.3 and
IgG.sub.4.
[0214] In one embodiment, the sequence of the humanized
immunoglobulin heavy chain variable region framework can be at
least about 65% identical to the sequence of the donor
immunoglobulin heavy chain variable region framework. Thus, the
sequence of the humanized immunoglobulin heavy chain variable
region framework can be at least about 75%, at least about 85%, at
least about 95%, or at least about 99% identical to the sequence of
the donor immunoglobulin heavy chain variable region framework. The
sequences of the heavy and light chain frameworks are known in the
art. Human framework regions, and mutations that can be made in a
humanized antibody framework regions, are known in the art (see,
for example, in U.S. Pat. No. 5,585,089).
[0215] Exemplary human antibodies LEN and 21/28 CL are of use in
providing framework regions. Exemplary light chain frameworks of
human MAb LEN have the following sequences:
TABLE-US-00001 FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 12) FR2:
WYQQKPGQPPLLIY (SEQ ID NO: 13) FR3:
GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 14) FR4: FGQGQTKLEIK
(SEQ ID NO: 15)
[0216] Exemplary heavy chain frameworks of human MAb 21/28' CL have
the following sequences:
TABLE-US-00002 FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ ID NO: 16)
FR2: WVRQAPGQRLEWMG (SEQ ID NO: 17) FR3:
RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 18) FR4: WGQGTLVTVSS.
(SEQ ID NO: 19)
These framework sequences are provided for example only; a
humanized antibody can include the human framework region from any
human monoclonal antibody of interest.
[0217] Fragments of the 5 kd106-13 D antibody (or other antibody
specific for an epitope of the OPN-5 kD fragment), a chimeric form
thereof, or a humanized form thereof are also encompassed by the
present disclosure. Antibodies, such as murine monoclonal
antibodies, chimeric antibodies, and humanized antibodies, include
full length molecules as well as fragments thereof, such as Fab,
F(ab').sub.2, and Fv which include a heavy chain and light chain
variable region and are capable of binding the epitope determinant
(e.g., amino acids 30-36 of SEQ ID NO:4). In some embodiments, the
antibodies fragments have the sequences for V.sub.L and V.sub.H
regions for the 5 kd106-13 D antibody. Fv antibodies are typically
about 25 kDa and contain a complete antigen-binding site with three
CDRs per each heavy chain and each light chain. To produce these
antibodies, the V.sub.H and the V.sub.L can be expressed from two
individual nucleic acid constructs in a host cell. If the V.sub.H
and the V.sub.L are expressed non-contiguously, the chains of the
Fv antibody are typically held together by noncovalent
interactions. However, these chains tend to dissociate upon
dilution, so methods have been developed to crosslink the chains
through glutaraldehyde, intermolecular disulfides, or a peptide
linker. Thus, in one example, the Fv can be a disulfide stabilized
Fv (dsFv), wherein the heavy chain variable region and the light
chain variable region are chemically linked by disulfide bonds.
[0218] In an additional example, the Fv fragments include V.sub.H
and V.sub.L chains connected by a peptide linker. These
single-chain antigen binding proteins (sFv) are prepared by
constructing a structural gene comprising DNA sequences encoding
the V.sub.H and V.sub.L domains connected by an oligonucleotide.
The structural gene is inserted into an expression vector, which
can be subsequently introduced into a host cell such as E. coli to
recombinantly express the antibody fragment. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFvs are known in
the art (see Whitlow et al., Methods: a Companion to Methods in
Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242:423,
1988; U.S. Pat. No. 4,946,778; and Pack et al., Bio/Technology
11:1271, 1993).
[0219] Antibody fragments can be prepared by proteolytic hydrolysis
of the antibody or by expression in E. coli of DNA encoding the
fragment. Antibody fragments can be obtained by pepsin or papain
digestion of whole antibodies by conventional methods. For example,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent, and optionally a blocking group for the sulfhydryl
groups resulting from cleavage of disulfide linkages, to produce
3.5S Fab' monovalent fragments. Alternatively, an enzymatic
cleavage using pepsin produces two monovalent Fab' fragments and an
Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No.
4,331,647, and references contained therein; Nisonhoff et al.,
Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119,
1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422,
Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10
and 2.10.1-2.10.4). Other methods of cleaving antibodies, such as
separation of heavy chains to form monovalent light-heavy chain
fragments, further cleavage of fragments, or other enzymatic,
chemical, or genetic techniques may also be used, so long as the
fragments bind to the antigen that is recognized by the intact
antibody.
[0220] One of skill will realize that conservative variants of the
antibodies can be produced. Such conservative variants employed in
antibody fragments, such as dsFv fragments or in scFv fragments,
will retain critical amino acid residues necessary for correct
folding and stabilizing between the V.sub.H and the V.sub.L
regions, and will retain the charge characteristics of the residues
in order to preserve the low pI and low toxicity of the molecules.
Amino acid substitutions (such as at most one, at most two, at most
three, at most four, or at most five amino acid substitutions) can
be made in the V.sub.H and the V.sub.L regions to increase yield.
Conservative amino acid substitution tables providing functionally
similar amino acids are well known to one of ordinary skill in the
art. The following six groups are examples of amino acids that are
considered to be conservative substitutions for one another:
[0221] 1) Alanine (A), Serine (S), Threonine (T);
[0222] 2) Aspartic acid (D), Glutamic acid (E);
[0223] 3) Asparagine (N), Glutamine (Q);
[0224] 4) Arginine (R), Lysine (K);
[0225] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0226] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0227] Thus, one of skill in the art can readily review the
sequences of an anti-OPN-5 kD monoclonal antibody (such as 5
kd106-13 D), locate one or more of the amino acids listed above,
identify a conservative substitution, and produce the conservative
variant using well-known molecular biology techniques. Generally,
conservative variants will bind the target antigen with an equal to
or greater efficiency than the parent monoclonal antibody.
[0228] Effector molecules, such as therapeutic, diagnostic, or
detection moieties can be linked to an antibody of interest, such
as an antibody that specifically binds OPN-5 kD (e.g., 5 kd106-13
D) using any number of means known to those of skill in the art. In
view of the large number of methods that have been reported for
attaching a variety of radiodiagnostic compounds, radiotherapeutic
compounds, labels (such as enzymes or fluorescent molecules),
drugs, toxins, and other agents to antibodies, one skilled in the
art will be able to determine a suitable method for attaching a
given agent to an antibody or fragment thereof. For example,
covalent and noncovalent attachment means can be used. The
procedure for attaching an effector molecule to an antibody varies
according to the chemical structure of the effector. Polypeptides
typically contain a variety of functional groups; such as
carboxylic acid (COOH), free amine (--NH.sub.2) or sulfhydryl
(--SH) groups, which are available for reaction with a suitable
functional group on an antibody to result in the binding of the
effector molecule. Alternatively, the antibody is derivatized to
expose or attach additional reactive functional groups. The
derivatization can involve attachment of any of a number of linker
molecules such as those available from Pierce Chemical Company,
Rockford, Ill. The linker can be any molecule used to join the
antibody to the effector molecule. The linker is capable of forming
covalent bonds to both the antibody and to the effector molecule.
Suitable linkers are well known to those of skill in the art and
include, but are not limited to, straight or branched-chain carbon
linkers, heterocyclic carbon linkers, or peptide linkers. Where the
antibody and the effector molecule are polypeptides, the linkers
may be joined to the constituent amino acids through their side
groups (such as through a disulfide linkage to cysteine) or to the
alpha carbon amino and carboxyl groups of the terminal amino
acids.
[0229] In some circumstances, it is desirable to free the effector
molecule from the antibody when the immunoconjugate has reached its
target site. Therefore, in these circumstances, immunoconjugates
will include linkages that are cleavable in the vicinity of the
target site. Cleavage of the linker to release the effector
molecule from the antibody may be prompted by enzymatic activity or
conditions to which the immunoconjugate is subjected either inside
the target cell or in the vicinity of the target site. When the
target site is a tumor, a linker which is cleavable under
conditions present at the tumor site (for example when exposed to
tumor-associated enzymes or acidic pH) may be used.
[0230] An antibody that specifically binds to the OPN-5 kD fragment
(e.g., 5 kd106-13 D or a humanized or chimeric form thereof or a
fragment thereof) can be labeled with one or more detectable agents
or labels. In some embodiments, labels are attached by spacer arms
of various lengths to reduce steric hindrance. Useful detectable
agents include electron-dense compounds, enzymes, fluorochromes,
haptens, and radioisotopes. Anti-OPN-5 kD antibodies (e.g., 5
kd106-13 D) can also be detected using secondary reagents with
specificity for mouse IgG. Useful detection agents include
fluorescent compounds, including fluorescein, fluorescein
isothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonyl
chloride, phycoerythrin, lanthanide phosphors, and the cyanine
family of dyes (such as Cy-3 or Cy-5) and the like. Bioluminescent
markers are also of use, such as luciferase, Green fluorescent
protein (GFP), Yellow fluorescent protein (YFP). An OPN-5 KD
antibody can also be labeled with enzymes that are useful for
detection, such as horseradish peroxidase, .beta.-galactosidase,
luciferase, alkaline phosphatase, glucose oxidase and the like.
When an antibody is labeled with a detectable enzyme, it can be
detected by adding additional reagents that the enzyme uses to
produce a reaction product that can be discerned. For example, when
the agent horseradish peroxidase is present the addition of
hydrogen peroxide and diaminobenzidine leads to a colored reaction
product, which is visually detectable. An antibody may also be
labeled with biotin, and detected through indirect measurement of
avidin or streptavidin binding. The avidin itself can also be
labeled with an enzyme or a fluorescent label. In one example, an
OPN-5 kD-specific antibody is labeled with electrodense particles,
such as a nanoparticle (for example a gold particle or a
semiconductor nanocrystal, such as a quantum dot (QDOT.RTM.)).
[0231] An antibody that specifically binds to the OPN-5 kD fragment
(e.g., 5 kd106-13 D or a humanized or chimeric form thereof or a
fragment thereof) can be labeled with a paramagnetic agent, such as
gadolinium. Antibodies can also be labeled with lanthanides (such
as europium and dysprosium), and manganese. Paramagnetic particles
such as superparamagnetic iron oxide are also of use as labels. An
antibody may also be labeled with a predetermined polypeptide
epitopes recognized by a secondary reporter (such as leucine zipper
pair sequences, binding sites for secondary antibodies, metal
binding domains, epitope tags).
[0232] Antibodies can also be labeled with a radiolabeled amino
acid, such as .sup.3H, .sup.14C, .sup.15N, .sup.35S, .sup.90Y,
.sup.99Tc, .sup.111In, .sup.125I, or .sup.131I. The radiolabel may
be used for diagnostic and/or therapeutic purposes. For instance,
the radiolabel may be used to detect OPN-5 kD expressing cells
(such as HCC cells) for example by x-ray or other diagnostic
techniques, such as positron emission tomography (PET) or magnetic
resonance imaging (MRI). Further, the radiolabel may be used
therapeutically as a toxin for tumors that express OPN (such as
HCC).
[0233] In some examples a therapeutic agent is linked to an
antibody that specifically binds to the OPN-5 kD fragment (e.g., 5
kd106-13 D or a humanized or chimeric form thereof or a fragment
thereof). Therapeutic agents include various drugs such as
vinblastine, daunomycin and the like, and effector molecules such
as cytotoxins for example native or modified Pseudomonas exotoxin
(e.g., see U.S. Pat. Nos. 4,892,827, 5,602,095, 5,608,039 and
5,512,658), ricin, abrin (e.g., see Funatsu et al., Agr. Biol.
Chem. 52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335,
1978), botulinum toxins A through F, or Diphtheria toxin (e.g., see
U.S. Pat. No. 5,792,458 and U.S. Pat. No. 5,208,021), ribonucleases
(e.g., see Suzuki et al., Nat. Biotech. 17:265-70, 1999),
encapsulating agents, (such as, liposomes) which themselves contain
pharmacological compositions, target moieties and ligands.
Exemplary toxins are well known in the art and many are readily
available from commercial sources (for example, Sigma Chemical
Company, St. Louis, Mo.). The choice of a particular therapeutic
agent depends on the particular target molecule or cell and the
biological effect desired to be evoked. Thus, for example, the
therapeutic agent may be an effector molecule that is cytotoxic
which is used to bring about the death of a particular target cell.
Conversely, where it is merely desired to invoke a non-lethal
biological response, a therapeutic agent can be conjugated to a
non-lethal pharmacological agent or a liposome containing a
non-lethal pharmacological agent.
[0234] The OPN-5 kD antibodies or antibody fragments disclosed
herein (e.g., 5 kd106-13 D) can be derivatized or linked to another
molecule (such as another peptide or protein). In general, the
antibodies or portion thereof is derivatized such that the binding
to the OPN-5 kD fragment is not affected adversely by the
derivatization or labeling. For example, the antibody can be
functionally linked (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (for example, a
bispecific antibody), a detection agent, a pharmaceutical agent, a
chemical group (such as polyethylene glycol (PEG), a methyl or
ethyl group, or a carbohydrate group for example to increase serum
half-life or to increase tissue binding of the antibody) and/or a
protein or peptide that can mediate association of the antibody or
antibody portion with another molecule (such as a streptavidin core
region or a polyhistidine tag).
[0235] One type of derivatized antibody is produced by crosslinking
two or more antibodies (of the same type or of different types,
such as to create bispecific antibodies). Suitable crosslinkers
include those that are heterobifunctional, having two distinctly
reactive groups separated by an appropriate spacer (such as
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional
(such as disuccinimidyl suberate). Such linkers are available from
Pierce Chemical Company, Rockford, Ill.
Nucleic Acid Molecules Encoding Antibodies
[0236] Nucleic acid molecules encoding the disclosed OPN-5 kD
antibodies (e.g., 5 kd106-13 D) or a chimeric or humanized form of
any of these antibodies or a fragment thereof can readily be
produced by one of skill in the art. Upon generation of a
monoclonal antibody, such as 5 kd106-13 D (see Example 9), the
amino acid sequence can be determined and a nucleic acid sequence
encoding the amino acid sequence can be engineered. In addition,
one of skill can readily construct a variety of clones containing
functionally equivalent nucleic acid molecules, such as nucleic
acid molecules which differ in sequence but which encode the same
effect or molecule ("EM") or antibody sequence. Thus, nucleic acids
encoding OPN-5 kD-specific antibodies (such as those specific for
the epitope EELNGAY, amino acids 30 to 36 of SEQ ID NO: 4),
conjugates and fusion proteins are provided herein.
[0237] Nucleic acid molecules encoding OPN-5 kD-specific antibodies
can readily be produced by one of skill in the art, using the amino
acid sequences provided herein, and the genetic code. In addition,
one of skill can readily construct a variety of clones containing
functionally equivalent nucleic acids, such as nucleic acids which
differ in sequence but which encode the same effector molecule or
antibody sequence. Thus, nucleic acids encoding OPN-5 kD-specific
antibodies, conjugates and fusion proteins are provided herein.
[0238] Nucleic acid sequences encoding the antibodies that
specifically bind OPN-5 kD (for example that are specific for the
EELNGAY epitope, amino acids 30 to 36 of SEQ ID NO: 4) can be
prepared by any suitable method including, for example, cloning of
appropriate sequences or by direct chemical synthesis by methods
such as the phosphotriester method of Narang et al., Meth. Enzymol.
68:90-99, 1979; the phosphodiester method of Brown et al., Meth.
Enzymol. 68:109-151, 1979; the diethylphosphoramidite method of
Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; the solid phase
phosphoramidite triester method described by Beaucage &
Caruthers, Tetra. Letts. 22(20):1859-1862, 1981, for example, using
an automated synthesizer as described in, for example,
Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168, 1984;
and, the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis produces a single stranded oligonucleotide. This can be
converted into double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill would recognize
that while chemical synthesis of DNA is generally limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
[0239] Exemplary nucleic acid molecules encoding sequences encoding
an antibody that specifically binds OPN-5 kD (e.g., 5 kd106-13 D)
can be prepared by cloning techniques. Examples of appropriate
cloning and sequencing techniques, and instructions sufficient to
direct persons of skill through many cloning exercises are found in
Sambrook et al., supra, Berger and Kimmel (eds.), supra, and
Ausubel, supra. Product information from manufacturers of
biological reagents and experimental equipment also provide useful
information. Such manufacturers include the SIGMA Chemical Company
(Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.), Pharmacia
Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo
Alto, Calif.), Chem Genes Corp., Aldrich Chemical Company
(Milwaukee, Wis.), Glen Research, Inc., GIBCO BRL Life
Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-Biochemika
Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San
Diego, Calif.), and Applied Biosystems (Foster City, Calif.), as
well as many other commercial sources known to one of skill in the
art.
[0240] Nucleic acid molecules can also be prepared by amplification
methods. Amplification methods include polymerase chain reaction
(PCR), the ligase chain reaction (LCR), the transcription-based
amplification system (TAS), the self-sustained sequence replication
system (3SR). A wide variety of cloning methods, host cells, and in
vitro amplification methodologies are well-known to persons skilled
in the art.
[0241] In one example, an antibody that specifically binds OPN-5 kD
(e.g., 5 kd106-13 D) is prepared by inserting the cDNA which
encodes a variable region from an antibody into a vector which
includes the cDNA encoding an EM, such as an enzyme or label. The
insertion is made so that the variable region and the EM are read
in frame so that one continuous polypeptide is produced. Thus, the
encoded polypeptide contains a functional Fv region and a
functional EM region. In one example, cDNA encoding an enzyme is
ligated to a scFv so that the enzyme is located at the carboxyl
terminus of the scFv. For example, a cDNA encoding horseradish
peroxidase or alkaline phosphatase, or a polypeptide marker of
interest can be ligated to a scFv so that the enzyme (or
polypeptide marker) is located at the amino terminus of the scFv.
In another example, the label is located at the amino terminus of
the scFv. In a further example, cDNA encoding the protein or
polypeptide marker is ligated to a heavy chain variable region of
an antibody, so that the enzyme or polypeptide marker is located at
the carboxyl terminus of the heavy chain variable region. The heavy
chain-variable region can subsequently be ligated to a light chain
variable region of the antibody using disulfide bonds. In a yet
another example, cDNA encoding an enzyme or a polypeptide marker is
ligated to a light chain variable region of an antibody, so that
the enzyme or polypeptide marker is located at the carboxyl
terminus of the light chain variable region. The light
chain-variable region can subsequently be ligated to a heavy chain
variable region of the antibody using disulfide bonds.
[0242] Once the nucleic acids encoding the anti-OPN-5 kD antibody
(e.g., 5 kd106-13 D), labeled antibody, or fragment thereof are
isolated and cloned, the protein can be expressed in a
recombinantly engineered cell such as bacteria, plant, yeast,
insect and mammalian cells using a suitable expression vector. One
or more DNA sequences encoding the antibody or fragment thereof can
be expressed in vitro by DNA transfer into a suitable host cell.
The cell may be prokaryotic or eukaryotic. The term also includes
any progeny of the subject host cell. It is understood that all
progeny may not be identical to the parental cell since there may
be mutations that occur during replication. Methods of stable
transfer, meaning that the foreign DNA is continuously maintained
in the host, are known in the art. Hybridomas expressing the
antibodies of interest (e.g., a hybridoma that produces 5 kd106-13
D) are also encompassed by this disclosure.
[0243] Polynucleotide sequences encoding the anti-OPN-5 kD antibody
(e.g., 5 kd106-13 D), labeled antibody, or functional fragment
thereof, can be operatively linked to expression control sequences.
An expression control sequence operatively linked to a coding
sequence is ligated such that expression of the coding sequence is
achieved under conditions compatible with the expression control
sequences. The expression control sequences include, but are not
limited to appropriate promoters, enhancers, transcription
terminators, a start codon (ATG) in front of a protein-encoding
gene, splicing signal for introns, maintenance of the correct
reading frame of that gene to permit proper translation of mRNA,
and stop codons.
[0244] The polynucleotide sequences encoding the OPN-5 kD-specific
antibody (e.g., 5 kd106-13 D), labeled antibody, or functional
fragment thereof can be inserted into an expression vector
including, but not limited to a plasmid, virus or other vehicle
that can be manipulated to allow insertion or incorporation of
sequences and can be expressed in either prokaryotes or eukaryotes.
Hosts can include microbial, yeast, insect and mammalian organisms.
Methods of expressing DNA sequences having eukaryotic or viral
sequences in prokaryotes are well known in the art. Biologically
functional viral and plasmid DNA vectors capable of expression and
replication in a host are known in the art.
[0245] Transformation of a host cell with recombinant DNA can be
carried out by conventional techniques as are well known to those
skilled in the art. Where the host is prokaryotic, such as E. coli,
competent cells which are capable of DNA uptake can be prepared
from cells harvested after exponential growth phase and
subsequently treated by the CaCl.sub.2 method using procedures well
known in the art. Alternatively, MgCl.sub.2 or RbCl can be used.
Transformation can also be performed after forming a protoplast of
the host cell if desired, or by electroporation.
[0246] When the host is a eukaryote, such methods of transfection
of DNA as calcium phosphate coprecipitates, conventional mechanical
procedures such as microinjection, electroporation, insertion of a
plasmid encased in liposomes, or virus vectors may be used.
Eukaryotic cells can also be cotransformed with polynucleotide
sequences encoding the antibody, labeled antibody, or functional
fragment thereof, and a second foreign DNA molecule encoding a
selectable phenotype, such as the herpes simplex thymidine kinase
gene. Another method is to use a eukaryotic viral vector, such as
simian virus 40 (SV40) or bovine papilloma virus, to transiently
infect or transform eukaryotic cells and express the protein (see
for example, Eukaryotic Viral Vectors, Cold Spring Harbor
Laboratory, Gluzman ed., 1982). One of skill in the art can readily
use an expression systems such as plasmids and vectors of use in
producing proteins in cells including higher eukaryotic cells such
as the COS, CHO, HeLa and myeloma cell lines.
[0247] Isolation and purification of recombinantly expressed
polypeptide can be carried out by conventional means including
preparative chromatography and immunological separations. Once
expressed, the OPN-5 kD-specific antibody (e.g., 5 kd106-13 D),
labeled antibody or functional fragment thereof can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, column chromatography, and
the like (see, generally, R. Scopes, Protein Purification,
Springer-Verlag, N.Y., 1982). Substantially pure compositions of at
least about 90 to 95% homogeneity are disclosed herein, and 98 to
99% or more homogeneity can be used for pharmaceutical purposes.
Once purified, partially or to homogeneity as desired, if to be
used therapeutically, the polypeptides should be substantially free
of endotoxin.
[0248] Methods for expression of single chain antibodies and/or
refolding to an appropriate active form, including single chain
antibodies, from bacteria such as E. coli have been described and
are well-known and are applicable to the antibodies disclosed
herein. See, Buchner et al., Anal. Biochem. 205:263-270, 1992;
Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science
246:1275, 1989 and Ward et al., Nature 341:544, 1989.
[0249] Often, functional heterologous proteins from E. coli or
other bacteria are isolated from inclusion bodies and require
solubilization using strong denaturants, and subsequent refolding.
During the solubilization step, as is well known in the art, a
reducing agent must be present to separate disulfide bonds. An
exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M
guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). Reoxidation of
the disulfide bonds can occur in the presence of low molecular
weight thiol reagents in reduced and oxidized form, as described in
Saxena et al., Biochemistry 9: 5015-5021, 1970, and especially as
described by Buchner et al., supra.
[0250] Renaturation is typically accomplished by dilution (for
example, 100-fold) of the denatured and reduced protein into
refolding buffer. An exemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M
L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.
[0251] As a modification to the two chain antibody purification
protocol, the heavy and light chain regions are separately
solubilized and reduced and then combined in the refolding
solution. An exemplary yield is obtained when these two proteins
are mixed in a molar ratio such that a 5-fold molar excess of one
protein over the other is not exceeded. Excess oxidized glutathione
or other oxidizing low molecular weight compounds can be added to
the refolding solution after the redox-shuffling is completed.
[0252] In addition to recombinant methods, the antibodies, labeled
antibodies and functional fragments thereof that are disclosed
herein can also be constructed in whole or in part using standard
peptide synthesis. Solid phase synthesis of the polypeptides of
less than about 50 amino acids in length can be accomplished by
attaching the C-terminal amino acid of the sequence to an insoluble
support followed by sequential addition of the remaining amino
acids in the sequence. Techniques for solid phase synthesis are
described by Barany & Merrifield, The Peptides: Analysis,
Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis,
Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc.
85:2149-2156, 1963, and Stewart et al., Solid Phase Peptide
Synthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984.
Proteins of greater length may be synthesized by condensation of
the amino and carboxyl termini of shorter fragments. Methods of
forming peptide bonds by activation of a carboxyl terminal end
(such as by the use of the coupling reagent
N,N'-dicylohexylcarbodimide) are well known in the art.
Use of OPN-5 kD Antibodies
[0253] The disclosure provides a method for detecting the OPN-5 kD
fragment in a biological sample, wherein the method includes
contacting a biological sample with an antibody that binds an OPN-5
kD epitope under conditions conductive to the formation of an
immune complex, and detecting the immune complex, to detect the
OPN-5 kD in the biological sample. The disclosure also provides a
method for treating a subject suspected of having an OPN expressing
tumor with OPN-5 kD specific antibodies, wherein the method
includes contacting a biological sample (in vitro or in vivo) with
an antibody that binds an OPN-5 kD epitope and inhibits or reduces
the binding of OPN to CD44 receptors (such methods are described
herein and more specifically in the section describing methods of
treatment and therapeutic compositions).
[0254] In one example, the detection of the OPN-5 kD fragment in
the sample indicates that the subject has a malignancy. In another
example, the detection of OPN-5 kD in the sample indicates that a
tumor in the subject is prone to metastasis.
[0255] In one example, the antibody that specifically binds OPN-5
kD is directly labeled with a detectable label. In another example,
the antibody that specifically binds OPN-5 kD (the first antibody)
is unlabeled and a second antibody or other molecule that can bind
the antibody that specifically binds OPN-5 kD is labeled. As is
well known to one of skill in the art, a second antibody is chosen
that is able to specifically bind the specific species and class of
the first antibody. For example, if the first antibody is a human
IgG, then the secondary antibody can be an anti-human-IgG. Other
molecules that can bind to antibodies include, without limitation,
Protein A and Protein G, which are available commercially.
[0256] Suitable labels for the antibody or secondary antibody are
described above, and include various enzymes, prosthetic groups,
fluorescent materials, luminescent materials, magnetic agents and
radioactive materials. Non-limiting examples of suitable enzymes
include horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase. Non-limiting examples
of suitable prosthetic group complexes include streptavidin/biotin
and avidin/biotin. Non-limiting examples of suitable fluorescent
materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein,
dansyl chloride or phycoerythrin. A non-limiting exemplary
luminescent material is luminol; a non-limiting exemplary a
magnetic agent is gadolinium, and non-limiting exemplary
radioactive labels include .sup.125I, .sup.131I, .sup.35S or
.sup.3H.
[0257] In an alternative example, the presence of the OPN-5 kD
fragment can be assayed in a biological sample by a competition
immunoassay utilizing OPN-5 kD standards labeled with a detectable
substance and an unlabeled antibody that specifically binds an
OPN-5 kD epitope. In this assay, the biological sample (such as
serum), the labeled OPN-5 kD standards and the antibody that
specifically binds OPN-5 kD are combined and the amount of labeled
OPN-5 kD standard bound to the unlabeled antibody is determined.
The amount of OPN-5 kD in the biological sample is inversely
proportional to the amount of labeled OPN-5 kD standard bound to
the antibody that specifically binds OPN-5 kD.
[0258] The immunoassays and methods disclosed herein can be used
for a number of purposes. In one example, the antibodies that
specifically bind OPN-5 kD are used to detect the OPN-5 kD in serum
obtained from a subject known to have or suspected of having a
tumor, such as HCC. Increased expression of OPN-5 kD is associated
with a tumor that expresses OPN, such as HCC, as well as increased
likelihood of metastasis of the tumor. Thus, the level of OPN-5 kD
can be used to diagnose, or determine the prognosis of, HCC in a
subject.
Example 1
OPN and MMP-9 are Co-Upregulated in HCC
[0259] This example describes methods used to demonstrate that OPN
and MMP-9 are co-upregulated in HCC.
[0260] Messenger RNA (mRNA) levels of OPN and MMP-9 were quantified
in normal and HCC tissue (obtained from human subjects) by
microarray analysis. As shown in FIG. 1A, expression of both OPN
and MMP-9 was significantly increased in primary HCC tumors and
their corresponding metastatic lesions as compared to
non-metastatic tumors (Ye et al., Nat. Med. 9:416-23, 2003). As
shown in FIG. 1B, up-regulated expression was significantly
correlated within each patient case (p=0.0013).
Example 2
OPN is a Substrate for MMP-9 Activity
[0261] This example describes methods used to demonstrate that
MMP-9 cleaves OPN into several fragments.
[0262] OPN cleavage assays were performed as follows. Human MMP-9
(Calbiochem, CA) was incubated at 37.degree. C. for 3 hours with
recombinant human OPN (R&D systems, MN) at an optimized ratio
(5:1 substrate:enzyme) in a buffered solution (200 mM NaCl, 5 mM
CaCl, 50 mM Tris-C1, pH 7.5). Digested OPN was analyzed by 16%
tris-glycine SDS-PAGE followed by colloidal coomassie blue staining
(SimplyBlue.TM., Invitrogen, CA). Separate cleavage reactions (2
.mu.g OPN) were transferred to PDVF membranes for amino-end
terminal microsequencing.
[0263] For western blot analysis, MMP-9-digested OPN samples and
whole cell lysates (50 .mu.g) were transferred to 0.22 um PVDF
membranes and probed with a polyclonal antibody generated to a
14-residue peptide just downstream of the residue 169 thrombin
cleavage site (KSKKFRRPDIQYPD; amino acids 169-183 of SEQ ID NO: 2)
(Abcam, MA). HEK-293 cell lysates were probed with an anti-FLAG
antibody (Sigma, Mo.). Immuno-reactive proteins were detected by
chemilluminescence (ECL, GE Healthcare Life Sciences, NJ).
[0264] Recombinant MMP-9 cleaved OPN at two predominant sites in
vitro, residues 166 and 210, resulting in 4 identified fragments:
OPN-34 kD (residues 1-166), OPN-32 kD (residues 167-314), OPN-5 kD
(residues 167-210), and OPN-24 kD (residues 211-314). Residue
numbering is based on the sequence shown in SEQ ID NO: 2. Two of
the fragments, OPN-32 kD and OPN-5 kD, were detected using an
antibody that recognizes residues 169-183 (FIG. 2A). The OPN-5 kD
fragment was distinct from the products of thrombin digestion and
could not be detected in cleavage reactions partially inhibited by
the concomitant addition of EDTA, a naturally-occurring inhibitor
of MMP-9 (tissue inhibitor of matrix metalloproteinase-1, or
TIMP-1), or the hydroxamate MMP-9 inhibitor I. Cleavage was
similarly inhibited by a monoclonal antibody to MMP-9. OPN-5 kD
formation appeared dependent on further cleavage of OPN-32 kD over
time (FIG. 2B).
[0265] Expression vectors were generated that included an OPN
fragment with its C-termini fused to a FLAG-tag and its N-termini
following the native OPN putative secretory leading 16 amino acid
peptide. Complementary DNA sequences encoding the wild-type OPN
protein (OPN-a) and three identified truncated protein sequences
described above (OPN-34 kD, OPN-5 kD, and OPN-24 kD) were cloned
into a CMV-promoter mammalian expression vector with a C-terminal
1.times.FLAG tag (pDest-490) (Gateway cloning system, Invitrogen,
CA). The 16-residue predicted signal peptide sequence
(MRIAVICFCLLGITCA, amino acids 1-16 of SEQ ID NO: 2) was fused to
the N-terminus of each construct following a Kozak translation
initiation sequence to ensure similar secretion. Entry clones were
verified by sequencing and gel electrophoresis. As shown in FIG.
3A, the native 16 amino acid signal sequence (indicated in black)
was included at the N-terminus of each of the 4 sequences. The RGD
domain was present in 2 of the 4 constructs (indicated in
white).
[0266] Effective expression was demonstrated in HEK 293 cells by
immunoblotting for the C-terminal FLAG tagged sequences as follows.
Secreted levels were also detected in conditioned media samples.
HEK 293 cells were transiently transfected (Lipofectamine 2000,
Invitrogen, CA) with 5 .mu.g plasmid DNA and harvested after 24
hours. Total protein cell lysates (50 .mu.g) were run on a 4-20%
SDS-PAGE gel, transferred to a nitrocellulose membrane and probed
with an anti-FLAG polyclonal antibody. As shown in FIG. 3B, all of
the constructs were expressed in HEK 293 cells.
Example 3
OPN-5 kD and MMP-9 Expression Correlate with Metastatic
Activity
[0267] This example describes methods used to demonstrate that
expression of the OPN-5 kD fragment (as well as full-length OPN)
and expression of MMP-9 correlated with metastatic potential.
[0268] Three human HCC cell lines (Hep3B, SMMC-7721, and MHCC-97)
of established inherent differences in invasiveness were
comparatively screened for OPN protein expression.
[0269] Single-cell clones of the low-metastatic HCC cell line
SMMC-7721 were produced by a limiting dilution cloning technique
(Morley et al., Exp. Hematol., 11:418-24, 1983). Briefly, early
passage parental cells were plated in 96-well plates (0.5
cell/well) in supplemented media (20% conditioned media) for two
weeks with bi-weekly media changes. Cells were propagated by
sequential splitting to generate large cell cultures for frozen
storage. The subclone most closely resembling the parental cell
line by morphology, growth, and a preliminary evaluation of
adhesion (see assay methods below), SMMC-7721-SC2, was used for
subsequent assays at early passage numbers (<10).
[0270] Total protein cell lysates (50 .mu.g) were run by 16%
SDS-PAGE, transferred to a nitrocellulose membrane, and probed with
a polyclonal OPN antibody (pAb8448) and a monoclonal actin antibody
(mAb1501). Recombinant human OPN and OPN-5 kD peptide samples were
run as controls (50 ng). As shown in FIG. 4A, endogenous OPN
expression corresponds to degree of metastatic potential, with the
highly-invasive cell line MHCC-97 expressing the most prominent
levels. Similarly, relative levels of the OPN-5 kD cleavage
fragment correlated with metastatic potential (FIG. 4A, left panel
and FIG. 4C). These results were reproduced using the OPN-5 kD
specific polyclonal antibody described in Example 8.
[0271] To determine the level of MMP-9 activity in the three human
HCC cell lines (Hep3B, SMMC-7721, and MHCC-97), gelatinase
activities in the cell lines were analyzed by zymography of
conditioned media samples. Gelatin zymography was performed as
follows. Cells were cultured for 48 hours in serum-free media. Cell
number-adjusted serum-free conditioned media harvested from the HCC
cell lines cultured to near-confluence were run on 10% tris-glycine
gels containing 0.1% gelatin (NOVEX, Invitrogen, CA). The separated
proteins were allowed to renature at 25.degree. C. for 1 hour (2.5%
Triton X-100) and develop at 37.degree. C. for 16 hours (50 mM
Tris, 0.2 M NaCl, 5 mM CaCl2, 0.02% Brij 35) followed by staining
(0.1% amido black). Pro-MMP-9, active MMP-9, and active MMP-2
standards were included as controls (0.5 ug).
[0272] As shown in FIG. 4B, the highest levels of gelatinases
(pro-MMP-9 and pro-MMP-2) were observed in MHCC-97 cell media.
Conversely, the two low-metastatic cell lines, Hep3B and SMCC-7721,
secreted nearly undetectable MMP-9 levels. These combined findings
implicate a correlated MMP-9/OPN abundance which may confer
differential levels of OPN-5 kD.
[0273] To measure the invasion of HCC cell lines, the following
methods were used. An equal number of cells (5.times.10.sup.4) was
seeded into the upper chambers of Matrigel-coated 8-um pore
membranes (HTSTM Fluoroblok tumor invasion system, BD Biosciences).
After 36 hours, the invaded cells were labeled with calcein
(37.degree. C., 1 hour) and fluorescence was measured (EX485,
EM530). As shown in FIG. 4C, the highest levels of invasion were
observed in MHCC-97 cells, while the two low-metastatic cell lines,
Hep3B and SMCC-7721, had lower levels of invasion.
Example 4
OPN Regions Differentially Modulate Cellular Adhesion and
Migration
[0274] This example describes methods used to demonstrate the
effects of OPN fragments on cellular adhesion and migration of HCC
cells.
[0275] Cellular adhesion was measured using the following methods.
SMMC-7721-SC2 cells were seeded into the wells of a 96-well
fluorescent-read plate in serum-free media (6.times.10.sup.4
cells/well) with human OPN (0-250 nM) with or without prior
digestion by MMP-9. Media and MMP-9 alone treated cells were
included as controls. A GRGDS blocking peptide (400 .mu.M) was
added to separate media control, intact OPN, and MMP-9 cleaved OPN
(250 nM) conditions. Non-adherent cells were removed with a PBS
wash (100 .mu.l) after one hour and adherent cells were quantified
by CyQuant GR dye incorporation (Molecular Probes, OR) and
detection at EX485, EM530.
[0276] As shown in FIG. 5A, increased SMMC-7721-SC2 adhesion was
observed in the presence of an exogenous MMP-9 cleaved OPN pool
versus intact OPN at >50 nM concentrations; addition of an
integrin-binding GRGDS peptide blocked both intact and cleaved
OPN-mediated adhesion.
[0277] To determine the affect of OPN fragments on adhesion, the
following methods were used. SMMC-7721-SC2 cells (1.times.10.sup.6)
were trypsinized (E-PET, Biosource, CA) and transfected with the
four OPN expression constructs shown in FIG. 3A and empty vector
DNA (5 .mu.g) (50-60% efficiency) using optimized conditions
(Nucleofection, AMAXA, MD) and plated to 6-well plates. After a
24-hour recovery period, the cells were trypsinized and seeded by
equal numbers (6.times.10.sup.4) into fluorescent-read 96-well
plates in serum-free media. Non-adherent cells were removed with a
PBS wash after 2-60 minutes and fluorescence was quantified at
EX485, EM530.
[0278] As shown in FIG. 5B, increased adhesion was observed in
response to all four of the OPN constructs at early time points
following transient over-expression (2-10 minutes). However, at
later time points (30-60 minutes), the OPN fragments containing the
RGD integrin binding motif (OPN-full-length and OPN-34 kD)
increased adhesion, whereas the non-RGD containing regions (OPN-5
kD and OPN-24 kD) decreased adhesion relative to controls (FIG.
5B).
[0279] Migration and invasion assays were performed as follows.
Transfected SMMC-7721-SC2 cells (transfected with 5 .mu.g plasmid
DNA) were allowed to recover in 5% serum containing media and were
re-counted prior to dilution in serum-free media. Cells were seeded
(5.times.10.sup.4) into the upper chambers of Matrigel coated 8 um
pore membranes (HTSTM Fluoroblok tumor invasion system, BD
Biosciences, MA) or uncoated 0.8 um pore membranes (HTSTM
Fluoroblok insert system, BD Biosciences, MA). Media containing
serum (5%) was added to the lower chambers as a chemotactic agent.
Blocking antibodies to either the integrin .alpha.V.beta.3 receptor
(clone LM609, Chemicon, CA) or a common determinant of the CD44
receptors (clone A020, Calbiochem, CA) were added to the upper
chambers (4 .mu.g/ml) at the time of cell seeding. Migrated cells
were labeled with calcein AM (Molecular Probes, OR) at 37.degree.
C. for 1 hour, and fluorescence was measured at 24, 36, and 48
hours (EX 490 nm, EM 530 nm).
[0280] As shown in FIG. 5C, full-length OPN and OPN-34 kD induced
SMMC-7721-SC2 migration whereas OPN-5 kD and OPN-24 kD expression
decreased migration relative to controls.
Example 5
OPN-5 kD Induces Cellular Invasion Via CD44, but Not Integrin
.alpha.V.beta.3, Receptors
[0281] This example describes methods used to determine the effect
of OPN fragments on invasion of HCC cells.
[0282] Cell invasion was measured as described in Example 4.
Briefly, each of the OPN vectors shown in FIG. 3A (5 .mu.g) were
separately transfected into SMMC-7721 cells (1.times.10.sup.6)
using optimized conditions (Nucleofection, AMAXA). An equal number
of cells (5.times.10.sup.4) was seeded into the upper chambers of
Matrigel-coated 8 um pore membranes (HTSTM Fluoroblok tumor
invasion system, BD Biosciences). After 36 hours, the invaded cells
were labeled with calcein at 37.degree. C. for 1 hour and
fluorescence was measured (EX485, EM530).
[0283] As shown in FIG. 6A, only the internal OPN-5 kD region of
OPN induced reproducible >2-fold increases in cellular invasion
in SMMC-7721-SC2 cells (p=0.001). Full-length OPN slightly
increased invasion, but not to the extent observed by OPN-5 kD.
Conversely, over-expression of the RGD-containing OPN-34 kD
fragment slightly decreased invasiveness compared to vector
transfected cells at 36 hours which was more noticeable at a longer
48 hour time point (p=0.005). No significant difference was
observed with OPN-34 kD or OPN-24 kD.
[0284] To determine the effect of blocking antibodies to the
integrin aV133 and CD44 receptors on the affect of invasion of HCC
cells expression OPN full-length or OPN-5 kD, integrin
.alpha.V.beta.3 (Chemicon, CA) or CD44 (Calbiochem, CA) blocking
antibodies were added (4 .mu.g/ml) to cells transiently expressing
OPN full-length and OPN-5 kD. Invading cells were measured at 36
hours following calcein incorporation as described above.
[0285] To determine the effect of varying concentrations of the
OPN-5 kD peptide on invasion of HCC cells, SMMC-7721 cells were
exposed to increasing concentrations of the OPN-5 kD peptide (0.4
.mu.M, 1 .mu.M and 2 .mu.M) and invading cells were measured after
36 hours following calcein incorporation as described above. A 44
residue peptide corresponding to the OPN-5 kD fragment:
LRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQD (SEQ ID NO: 4), was
synthesized and purified >97% by HPLC (EZBiolabs, IN). A GRADSP
peptide of no known biological function was tested in parallel as a
control. Cells transiently transfected with the OPN-5 kD plasmid
were included for comparison.
[0286] As shown in FIG. 6B, addition of a blocking antibody that
recognizes a common determinant of CD44 receptors significantly
reduced OPN-5 kD induced invasion (p=0.0005), whereas a blocking
antibody to the integrin .alpha.V.beta.3 receptor had no
significant effect. Similar results were observed when a synthetic
peptide corresponding to OPN-5 kD was used in a
concentration-dependent manner (FIG. 6C).
[0287] These results indicate the OPN-5 kD peptide induced cellular
invasion is contingent upon CD44 receptor interaction. Similar
patterns in adhesive, migratory, and invasive responses were
reproduced in the other low-metastatic HCC cell line Hep3B.
Example 6
Fragments of OPN-5 kD reduce OPN-5 kD-induced cellular invasion
[0288] This example describes methods used to identify a minimum
region responsible for the observed OPN-5 kD-mediated cellular
invasion and the identification of fragments that reduce OPN
induced cellular invasion.
[0289] A 44 residue peptide corresponding to the OPN-5 kD fragment
(SEQ ID NO: 4), was synthesized and purified >97% by HPLC
(EZBiolabs, IN). A series of short peptides spanning the region of
residues 167-210 of OPN (SEQ ID NO: 2) were generated as follows.
Eight small overlapping peptides spanning the OPN-5 kD peptide were
similarly synthesized to >75% purity (p1=LRSKSKKFRR, amino acids
167-176 of SEQ ID NO: 2; p2=KKFRRPDIQY, amino acids 172-181 of SEQ
ID NO: 2; p3=PDIQYPDATD, amino acids 177-186 of SEQ ID NO: 2;
p4=PDATDEDITS, amino acids 182-191 of SEQ ID NO: 2; p5=EDITSHMESE,
amino acids 187-196 of SEQ ID NO: 2; p6=HMESEELNGA, amino acids
192-201 of SEQ ID NO: 2; p7=ELNGAYKAIP, amino acids 197-206 of SEQ
ID NO: 2; and p8=YKAIPVAQD amino acids 202-210 of SEQ ID NO: 2)
(EZBiolabs, IN). The lyophilized peptides were resuspended in PBS
and applied with cells to the upper wells of Matrigel invasion
chambers at equal molar concentrations (2 .mu.M). Invading cells
were measured after 36 hours following calcein incorporation as
described in the examples above.
[0290] Exogenous treatment of these short peptides to SMMC-7721 SC2
cells at equal molar concentrations did not increase cellular
invasion as compared to OPN-5 kD or a control peptide of no known
ability to bind cell surface receptors (GRADSP). However, three
peptides (p3=PDIQYPDATD, SEQ ID NO: 5; p6=HMESEELNGA, SEQ ID NO: 6;
and p7=ELNGAYKAIP, SEQ ID NO: 7) had a suppressive effect relative
to media alone treated cells (p<0.05) (FIG. 7A).
[0291] Upon separate addition of p3, p6, and p7 in conjunction with
OPN-5 kD (1:1 molar concentrations, 2 .mu.M each), a partial
reduction in invasion was observed (p<0.002) which contrasted
the insignificant effect of another peptide, p2 (FIG. 7B). The
combined addition of p3, p6, and p7 completely abolished the OPN-5
kD-mediated invasion down to the levels of media controls
(p=2.times.10.sup.-5).
Example 7
OPN-c is Highly Expressed in Metastatic HCC
[0292] This example describes methods used to identify the OPN
splice variants expressed in HCC.
[0293] Quantitative RT-PCR was performed as follows. RNA was
isolated from HCC primary metastatic tumors (n=17), primary
non-metastatic tumors (n=15), and paired non-cancerous tissues
collected >2 cm from the lesions, using Trizol (Invitrogen, CA).
Normal liver tissue was used as a reference pool (n=8). Total RNA
was quality assessed prior to reverse-transcription (cDNA archive
kit, Applied Biosystems, CA). Primers were designed upstream and
downstream from unique reporter sequences for OPN-a
(TCTCCTAGCCCCACAGAATGCTGTG (25-mer, SEQ ID NO: 10), 66-bp final
amplicon) and OPN-c (AGGAAAAGCAGAATGCTGTGTCCTC (25-mer, SEQ ID NO:
11), 92-bp final amplicon) (TaqMan gene expression assays, Applied
Biosystems, CA). CT threshold values were normalized to 18S rRNA
(Applied Biosystems, CA) and data were presented as fold changes
relative to the pooled normal liver samples.
[0294] Increased mRNA levels corresponding to both the wild-type
OPN-a and the variant OPN-c were detected in metastatic HCC tumor
samples compared with non-metastatic HCC (FIG. 8A). This effect was
notably greater for OPN-c (p=0.00074 vs. p=0.019 for OPN-a).
Further, increased OPN-c levels were detected in non-cancerous
tissue surrounding metastatic lesions but were not observed for
OPN-a, indicating the involvement of the surrounding stroma in a
differential expression of this OPN variant and conditions favoring
metastasis (FIG. 8B). A similar difference in OPN-c transcript
levels was reflected in the degree of metastatic potential in three
highly metastatic HCC cell lines evaluated relative to two
low-metastatic cell lines following normalization to primary
hepatocytes.
[0295] These results indicate greater mRNA level expression of
OPN-c than the wild-type OPN-a in metastatic HCC. The adjacent
non-cancerous tissues of the metastatic tumor samples are also
higher in OPN-c but not OPN-a levels. Histological analysis of this
surrounding tissue indicates a predominance of Kupffer-like cells,
likely indicating the contribution of macrophages in the tumor cell
environment to this observed transcript level response. Thus, these
results indicate a macrophage contribution to MMP-9 levels in the
tumor cell microenvironment. Such tumor-stromal cell interactions
and cross-talk via soluble cytokines and other factors appear to
play a role in malignant cell invasion in a multi-cellular tumor
microenvironment.
Example 8
Polyclonal Antibody Generation
[0296] This example describes methods used to generate polyclonal
antibodies to the OPN-5 kD peptide.
[0297] Un-conjugated OPN-5 kD synthetic peptide (synthesized as
described in Example 6) was injected into 2 rabbits (3.times.500 ug
doses), followed by a boost injection (100 .mu.g) at 84 days
following initial immunization (Animal Pharm Services, CA).
Post-boost bleedings were conducted every 2 weeks for 6 months and
immuno-specific antibodies were affinity purified using an OPN-5 kD
peptide column (Affigel 15, Biorad, CA). The resulting IgG antibody
was characterized using standard immunoblotting and
immunoprecipitation methods.
Example 9
Monoclonal Antibody Generation
[0298] This example describes methods used to generate monoclonal
antibodies to the OPN-5 kD peptide.
[0299] Standard methods were used to produce monoclonal antibodies
that were specific for OPN-5 kD (for example see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New
York, 1988). SEQ ID NO: 4 was injected into mice, followed by boost
injections, to produce monoclonal antibodies. Murine hybridomas
were produced. Antibody-producing clones were identified by
detection of antibody in the supernatant fluid by Western blotting.
One selected positive clone (5 kd106-13 D) was expanded and the
monoclonal antibody product harvested.
[0300] Hybridoma 5 kd106-13 D had the highest specificity and
sensitivity to binding the OPN-5 kD fragment. As shown in FIG. 9, 5
kd106-13 D antibody detects the OPN-5 kD fragment with high
specificity, and also has some reactivity to full-length OPN. It is
thought that full length OPN is bound to the blotting membrane in a
manner that partially blocks access of the 5 kd106-13 D monoclonal
antibody to the epitope within the OPN-5 kD epitope sequence
contained in the full length molecule.
[0301] The epitope of the monoclonal antibody produced by hybridoma
5 kd106-13 D is EELNGAY (amino acids 30-36 of SEQ ID NO: 4).
Example 10
Humanization and Production of an scFv
[0302] This example provides methods that can be used to determine
the CDR amino acid sequences of the monoclonal antibody 5 kd106-13
D described in Example 9. CDR amino acid sequences from monoclonal
antibody 5 kd106-13 D are used for humanization and construction of
recombinant scFv and scFv.sub.2 fragments. Protein-based and
cell-based assays have been used extensively for the purpose of
evaluating engineered antibodies (reviewed by Qu et al., Methods.
36:84-95, 2005).
[0303] Competitive cell-based binding assays are developed to
compare the antigen binding capabilities of engineered antibodies
with those of the parental mouse monoclonal antibodies. For initial
assays, human HCC cells are used as a source of target cells. For
these assays, unlabeled engineered antibodies are used as a
competitor of antigen binding by phycoerythrin (PE)-labeled
parental antibodies. Briefly, human HCC cells are plated at
1.times.10.sup.5 cells/well in a 96-well plate (100 uL/well). A
constant amount of PE-labeled parental antibody (10 nM) is mixed
with varying concentrations of unlabeled parental or engineered
antibodies (0.2-1,000 nM) and added to each well (100 uL/well),
with each experimental condition set up in triplicate. Plates are
preblocked to prevent binding of PE-conjugated antibody to the
plate surface (phosphate buffered saline (PBS), 0.05% Tween 20, and
5% fetal calf serum (FCS) for 2 hours at room temperature). After
adding cells plus antibodies, the plates are incubated on ice with
gentle mixing for 2 hr. Plates are then centrifuged and washed five
times to eliminate unbound PE-labeled antibody and evaluated for PE
signal using a fluorescence plate reader. The fluorescence
associated with cells is plotted versus the concentration of
unlabeled antibodies, yielding competitive inhibition curves.
Successful engineering results in similar curves for the engineered
and parental antibodies. Competitive radio-immunoassays can also be
used as an alternative for this determination.
Example 11
Detection of OPN-5 kD Using Antibodies
[0304] This example describes methods that can be used to detect
the OPN-5 kD fragment using OPN-5 kD specific antibodies, such as
those specific for the epitope EELNGAY (amino acids 30-36 of SEQ ID
NO: 4), for example antibodies produced by hybridoma 5 kd106-13 D
(see Example 9). For example, OPN-5 kD can be detected in a
biological sample, such as a tumor sample or blood sample (or
fraction thereof) to facilitate disease diagnosis in patients not
previously diagnosed with disease, confirmation of diagnosis in
patients with tentative diagnosis, or disease monitoring in
patients undergoing treatment for disease. In some examples, the
levels of OPN-5 kD are quantified. For example, such methods can be
used to diagnose the presence of an OPN-expressing tumor, such as
HCC or metastatic HCC, provide a prognosis of a subject having such
a tumor (for example determine the likelihood that a patient will
likely develop a metastasis), or combinations thereof.
[0305] In one example, serum specimens are evaluated from patients
with HCC as well as normal subjects who do not have HCC or other
cancer for the presence of the OPN-5 kD fragment bound by
monoclonal antibody 5 kd106-13 D (or other monoclonal antibody
specific for the epitope EELNGAY, amino acids 30-36 of SEQ ID NO:
4). Serum specimens are evaluated from 20-50 HCC patients using
Western blot analyses, direct ELISA, or sandwich ELISA. Antibody
reactivity with serum specimens from normal donors (negative
controls) is also assessed. Blood samples (10 mL) for preparation
of serum is obtained and serum is aliquoted and stored at
-80.degree. C. until used. In some examples, serum is also analyzed
for the presence of full-length OPN.
[0306] In some examples, full-length OPN (e.g., SEQ ID NO: 2) is
removed from the serum prior to detection of OPN-5 kD, for example
using filtration to remove higher molecular weight proteins (e.g.,
full-length OPN) and allowing lower molecular weight proteins
(e.g., OPN-5 kD) to pass through. In some examples there is no
fluid dilution factor so concentration of OPN-5 kD in the filtrate
is the same as it was in the unfractionated serum. For example,
ultrafiltration methods can be used to physically separate the
OPN-5 kD fragment from OPN.
[0307] In some examples, competition ELISA is used to detect the
OPN-5 kD fragment in serum using an ultra sensitive
electrochemiluminescence detection system. Competition assays are
well-known in the art. In one example, the 5 kd106-13 D monoclonal
antibody is used as the solid phase capture reagent and
ruthenium-labeled OPN-5 kD fragment as the equilibrium detection
reagent. Addition of the serum or other test sample competes for
the binding to the capture reagent. This results in a reduction of
signal as unlabeled OPN-5 kD fragment competes with the
ruthenium-labeled OPN-5 kD fragment. Ruthenium is caused to
chemiluminesce by electrical current in the microtiter plate (e.g.,
provided by Meso Scale Discovery, Gaithersburg, Md.) and the
resultant signal is measured (e.g., by CCD camera).
[0308] It is expected that the levels of the OPN-5 kD fragment in
serum from normal volunteers will be significantly decreased
relative to patients with hepatocellular carcinoma (and conversely,
the levels of the OPN-5 kD fragment in serum from HCC patients will
be significantly increased (for example at least 1.5-fold, at least
2-fold or even at least 5-fold) relative to normal patients), thus
demonstrating the utility of monoclonal antibodies provided herein
to diagnose or prognose an OPN-expressing tumor such as HCC.
Example 12
OPN-5 kD Specific Antibodies Reduce OPN-5 kD-Induced Cellular
Invasion
[0309] This example describes methods that can be used to
demonstrate that antibodies specific for OPN-5 kD, such as those
described in Examples 8 and 9 can inhibit cellular invasion in
vitro.
[0310] OPN-5 kD antibodies (e.g., 5 kd106-13 D or other antibody
specific for the same epitope) are suspended in PBS and applied
with cells to the upper wells of Matrigel invasion chambers at
equal molar concentrations (2 .mu.M). Invading cells are measured
after 36 hours following calcein incorporation as described in the
examples above (see Examples 4-6).
[0311] Therapeutic antibodies are identified as those that when
added at equal molar amounts with OPN-5 kD do not increase cellular
invasion as compared to OPN-5 kD (alone) or a control peptide of no
known ability to bind cell surface receptors (GRADSP). Therapeutic
antibodies are also identified as those that decrease migration as
compared to the controls.
Example 13
Treatment of HCC in an Animal Model Using OPN5-kD Antibodies
[0312] This example describes methods than can be used to
demonstrate that a therapeutic composition that includes one or
more OPN-5 kD specific antibodies can be used to treat HCC (such as
metastatic HCC) in an animal model. One skilled in the art will
appreciate that similar methods can be used to treat other
OPN-overexpressing tumors.
[0313] Monoclonal OPN-5 kD specific antibodies, such as those
described in Example 9, for example 5 kd106-13 D or other
monoclonal antibody specific for the epitope EELNGAY (amino acids
30-36 of SEQ ID NO: 4), will be effective in blocking the growth
and invasiveness of HCC cancer cells in vivo. The effectiveness of
such antibodies in blocking the growth of HCC tumors is tested
using subcutaneous implantation of HCC tumor cells in mice as a
model. The primary advantages of subcutaneous models are the ease
of implantation and subsequent monitoring of tumor size.
5.times.10.sup.5 HCC cells are inoculated subcutaneously into the
right craniolateral thorax (axilla) using aseptic technique. Tumors
are measured every 34 days using vernier calipers until they reach
a volume of 0.2-0.3 cm.sup.3. At that time, the mice are divided
into four groups (8-10 animals each): Group 1 (vehicle control),
Groups 2-4 are treated with 0.1, 1.0, or 10 mg/kg a particular test
antibody (e.g., 5 kd106-13 D), administered intraperitoneally,
twice a week. The mice are then monitored every 3 days to measure
tumor volume (with vernier calipers), body weight, and life span.
After no greater than 60 days past implantation, the animals are
sacrificed and postmortem evaluations of tumorigenesis, including
measurement and weight of implanted tumors and proximal lymph
nodes, macroscopic evaluation of soft tissues for tumors (lymph
nodes and lung), and formalin fixation of the primary tumor and
tissues, are performed. The tissues are evaluated by
immunohistochemistry using OPN specific antibodies to determine the
level of OPN expression in the tumors. In particular, tumor cells
that escape treatment are studied to determine whether they have
low levels of OPN expression.
[0314] While subcutaneous implantation is a popular and valuable
method for modeling tumor cell growth, differences between the
microenvironment of the skin and liver can cause rather dramatic
differences in cell behavior. For example, HCC may not metastasize
when implanted subcutaneously whereas intrahepatic implantation
(orthotopic) may facilitate metastasis. Thus, HCC cells are
implanted in the liver by exposing the liver via laparotomy and
inoculating tumor cells into liver using a surgical microscope.
After seven days, the mice begin receiving treatment with antibody,
as described above, and the animals are sacrificed no later than 60
days after implantation (or if the animals become moribund). The
tumors are palpated at 3-5 day intervals, at which time data on
tumor size, animal weight, and survival are collected. Post-mortem
evaluations are also performed as described above, with emphasis
upon the effect of antibody upon metastatic potential (to lungs and
regional lymph nodes). The monoclonal antibodies are believed to
block the primary tumor and metastatic potential of HCC cells in a
dose-dependent manner.
[0315] To minimize identification of strain or clonal-specific
effects, identical analyses using other model systems can be
employed.
[0316] One skilled in the art will appreciate other models for
liver cancer can be used, such as s.c. injection of MHCC97 cells
with 100% penetrance to develop lung metastasis in 5 weeks in nude
mice (see Ye et al., Nat. Med., 10:416, 2003). OPN-5 kD-specific
antibodies are then administered and mice screened as described
above. In some examples metastatic tumor cells are administered in
the tail-vein injection and lung metastasis monitored.
Example 14
Treatment of HCC in an Animal Model Using Fragments of OPN-5 kD
[0317] This example describes methods than can be used to
demonstrate that a therapeutic composition that includes one or
more OPN-5 kD peptide fragments, such as a peptide 5 to 60 amino
acids in length that has at least 5 contiguous amino acids of OPN-5
kD (e.g., SEQ ID NO: 4), can be used to treat HCC (such as
metastatic HCC) in an animal model. One skilled in the art will
appreciate that similar methods can be used to treat other
OPN-expressing tumors.
[0318] Mice recognized in the art as a model of HCC can be used.
For example, the nude mice model LCI-D20 (see Tang et al., J.
Cancer Res. Clin. Oncol. 130:187-96, 2004) or the model described
in Ye et al. (Nat. Med., 10:416, 2003) can be used. Mice are
administered at least 1 .mu.g of one or more OPN-5 kD peptide
fragments 5 to 60 amino acids in length having at least 5
contiguous amino acids of OPN-5 kD (e.g., SEQ ID NO: 4), such as
any of SEQ ID NOS: 5-8, intravenously or at least 100 .mu.g (such
as 1 mg) intramuscularly. These peptides can be formulated with an
inert diluent or with a pharmaceutically acceptable carrier. If
desired, other therapeutic molecules can also be administered, such
as one or more anti-neoplastic agents. The therapeutic compositions
can be administered in a single dose delivery, via continuous
delivery over an extended time period, in a repeated administration
protocol (for example, by a, daily, weekly, or monthly repeated
administration protocol). In one example, mice receive at least
weekly doses of the peptide over at least 6 months. Control mice
can receive no peptide (such as an injection that only includes the
pharmaceutical carrier).
[0319] Following the administration of one or more OPN-5 kD peptide
fragments, mice are monitored for tumor treatment, such as
regression or reduction in metastatic lesions. In particular
examples, mice are analyzed one or more times, starting 7 days
following treatment. Mice can be monitored using any method known
in the art. For example, diagnostic imaging can be used (such as
x-rays, CT scans, MRIs, ultrasound), as well as analysis of
biological samples (for example analysis of blood, tissue biopsy,
or other biological samples), such as analysis of the type of cells
present, or analysis for a particular tumor marker.
[0320] A reduction in the number or size of tumors, or a prolonged
survival time, in the experimental mice, as compared to the control
mice, indicates that the one or more OPN-5 kD peptide fragments
have an anti-neoplastic therapeutic effect.
Example 15
Treatment of HCC in Humans with Anti-OPN-5 kD or Fragments of OPN-5
kD
[0321] This example describes particular methods that can be used
to treat a primary or metastatic HCC tumor in humans by
administration of (1) an antibody specific for OPN-5 kD (such as 5
kd106-13 D or another monoclonal antibody that recognizes the same
epitope), (2) one or more OPN-5 kD peptide fragments, such as a
peptide 5 to 60 amino acids in length that has at least 5
contiguous amino acids of OPN-5 kD (e.g., SEQ ID NO: 4), or both
(1) and (2). Although particular methods, dosages, and modes of
administrations are provided, one skilled in the art will
appreciate that variations can be made without substantially
affecting the treatment. One skilled in the art will appreciate
that similar methods can be used to treat other OPN-expressing
tumors.
[0322] Therefore human patients are treated intravenously with at
least 1 .mu.g (such as 1-100 .mu.g) of one or more peptides that
include or consist of the sequence shown in any of SEQ ID NOS: 5-8,
for example for a period of at least 6 months, at least one year,
at least 2 years, or at least five years. Administration of the
peptides can be used in conjunction with normal cancer therapy (for
example rather than replacing the therapy). Thus the therapeutic
peptides can be added to the usual and customary chemotherapy,
surgery and/or radiation treatments conventionally used for the
particular tumor type, such as HCC. Administration of the
therapeutic peptides can be continued after chemotherapy and
radiation therapy was stopped and can be taken long term (for
example over a period of months or years).
[0323] Similarly, human patients are treated intravenously with at
least 1 .mu.g of 5 kd106-13 D (or a humanized form, chimeric form,
or fragment thereof, or another monoclonal antibody that recognizes
the same epitope), for example for a period of at least 6 months,
at least one year, at least 2 years, or at least five years.
Administration of the antibodies can be used in conjunction with
normal cancer therapy (for example rather than replacing the
therapy). Thus the therapeutic antibodies can be added to the usual
and customary chemotherapy, surgery and/or radiation treatments
conventionally used for the particular tumor type, such as HCC.
Administration of the therapeutic antibodies can be continued after
chemotherapy and radiation therapy was stopped and can be taken
long term (for example over a period of months or years).
[0324] Briefly, the method can include screening subjects to
determine if they have HCC, such as primary or metastatic HCC.
Subjects having HCC are selected. In one example, subject having
increased levels of the OPN-5 kD fragment in their serum are
selected. In a clinical trial, half of the subjects would follow
the established protocol for treatment of HCC (such as a normal
chemotherapy/radiotherapy/surgery regimen). The other half would
follow the established protocol for treatment of the tumor (such as
a normal chemotherapy/radiotherapy/surgery regimen) in combination
with administration of the therapeutic peptides or antibodies
described above. In some examples, the tumor is surgically excised
(in whole or part) prior to treatment with the therapeutic peptides
or antibodies.
Screening Subjects
[0325] In particular examples, the subject is first screened to
determine if they have HCC. Examples of methods that can be used to
screening for HCC include a combination of ultrasound, tissue
biopsy, and examination of alpha-feta protein (AFP) levels, wherein
serum AFP levels greater than 20 ng/ml or greater than 400 ng/ml
and a positive imaging result indicate that the subject has
HCC.
[0326] In some examples, the tumor is analyzed to determine if it
overexpresses OPN, MMP-9, or both, wherein the presence of such
overexpression indicates that the tumor can be treated with the
disclosed therapies. For example serum can be screened for the
presence of OPN-5 kD, and the tumor can be screened for OPN-c (and
in some examples also OPN-a) expression.
[0327] However, such pre-screening is not required prior to
administration of the therapeutic peptides or antibodies described
herein.
Pre-Treatment of Subjects
[0328] In particular examples, the subject is treated prior to
administration of therapeutic peptides or antibodies described
herein. However, such pre-treatment is not always required, and can
be determined by a skilled clinician. For example, the tumor can be
surgically excised (in total or in part) prior to administration of
one or more therapeutic peptides or antibodies described herein. In
addition, the subject can be treated with an established protocol
for treatment of the particular tumor present (such as a normal
chemotherapy/radiotherapy regimen).
Administration of Therapeutic Compositions
[0329] Administration can be achieved by any method known in the
art, such as oral administration, inhalation, or inoculation (such
as intramuscular, ip, or subcutaneous). In some examples, the
therapeutic composition includes one or more of SEQ ID NOS: 5-7 or
antibody 5 kd106-13 D (or a humanized form, chimeric form, or
fragment thereof).
[0330] The amount of one or more therapeutic peptides or antibodies
administered is sufficient to treat a subject having HCC. An
effective amount can being readily determined by one skilled in the
art, for example using routine trials establishing dose response
curves. In addition, particular exemplary dosages are provided
above. The therapeutic compositions can be administered in a single
dose delivery, via continuous delivery over an extended time
period, in a repeated administration protocol (for example, by a,
daily, weekly, or monthly repeated administration protocol).
[0331] In one example, therapeutic compositions that include an
antibody specific for OPN-5 kD (such as 5 kd106-13 D or another
monoclonal antibody that recognizes the same epitope) or one or
more OPN-5 kD peptide fragments, such as a peptide 5 to 60 amino
acids in length that has at least 5 contiguous amino acids of OPN-5
kD (e.g., SEQ ID NO: 4) are administered iv to a human. As such,
these compositions may be formulated with an inert diluent or with
an pharmaceutically acceptable carrier.
Assessment
[0332] Following the administration of one or more therapies,
subjects having a tumor (for example HCC) can be monitored for
tumor treatment, such as regression or reduction in metastatic
lesions. In particular examples, subjects are analyzed one or more
times, starting 7 days following treatment
[0333] Subjects can be monitored using any method known in the art.
For example, diagnostic imaging can be used (such as x-rays, CT
scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic
examination), as well as analysis of biological samples from the
subject (for example analysis of blood, tissue biopsy, or other
biological samples), such as analysis of the type of cells present,
or analysis for a particular tumor marker. In one example, if the
subject has a metastatic HCC, assessment can be made using
ultrasound, MRI, or CAT scans, analysis of the type of cells
contained in a tissue biopsy, and AFP levels.
Example 16
Evaluation Following Treatment
[0334] During or following therapeutic treatment (such as that
described in Example 15), subjects can be monitored for the
response of their tumor(s) to the therapeutic peptides or
antibodies.
[0335] Subjects can receive a complete physical evaluation, CBC,
acute care, hepatic and mineral panels and appropriate evaluations
of all evaluable lesions (for example by x-ray, MRI, CT scan,
ultrasound) are obtained every 6-12 weeks during the first six
months of therapy and if stable, every 3-6 months thereafter. Other
evaluations can be performed as indicated by symptoms or physical
findings. For example, surveillance CT of the chest, abdomen and
pelvis can be obtained with at least every other assessment and as
indicated by symptoms or physical findings.
Example 17
Additional Treatment
[0336] In particular examples, if subjects are stable or have a
minor, mixed or partial response to treatment, they can be
re-treated after re-evaluation with the same schedule and
preparation of peptide or antibody that they previously received
for up to a year of total therapy.
[0337] A mixed response is the shrinkage of some lesions but an
increase in others. Subjects with mixed responses may only receive
treatment for an additional 2-3 months without showing true disease
stability or a bona fide minor or major response (i.e., no further
progression). Two re-treatment cycles can be given following a
complete response.
Example 18
Diagnosis of HCC in Humans
[0338] This example describes particular methods that can be used
to diagnose or prognose HCC (such as metastatic HCC) in a human
subject. However, one skilled in the art will appreciate that
similar methods can be used. In some examples, such diagnosis is
performed before treating the subject (for example as described in
Example 15).
[0339] Biological samples are obtained from the subject. If blood
or a fraction thereof (such as serum) is used 0.1-10 ml of blood is
collected. Serum can either be used directly or fractionated using
filter cut-offs to remove high molecular weight proteins (such as
full-length OPN). If desired, the serum can be frozen and thawed
before use. If a tissue biopsy sample is used, 1-100 .mu.g of
tissue is obtained, for example using a fine needle aspirate RNA is
isolated from the tissue using routine methods (for example using a
commercial kit).
[0340] In one example, OPN-5 kD protein levels are determined in a
serum sample obtained from the subject. The serum sample described
above is incubated with the antibody described in Example 9 (5
kd106-13 D) for a time sufficient for the antibody to bind to OPN-5
kD in the serum. The OPN-5 kD/antibody complexes are detected, for
example using an ELISA. Alternatively, the serum sample is
subjected to 16% SDS-PAGE, and transferred to a membrane (such as
nitrocellulose), which is probed with the 5 kd106-13 D antibody.
The antibody/OPN-5 kD complexes can be detected with a secondary
labeled antibody, or by observing the appropriated sized protein on
the gel. The relative amount of OPN-5 kD/antibody complexes in the
serum sample from the subject can be compared to a reference value,
such as a relative amount of OPN-5 kD/antibody complexes present in
a serum sample from a subject not having a tumor, wherein the
presence of significantly more OPN-5 kD/antibody complexes in the
test sample as compared to the reference sample (such as an
increase of at least 2-fold, at least 3-fold, or at least 5-fold)
indicates that the subject has HCC, has metastatic HCC, has a poor
prognosis, or combinations thereof.
[0341] In one example, OPN-c mRNA expression levels are determined
in a tumor sample obtained from the subject and in a tissue sample
adjacent to (but not including) the tumor. cDNA is generated from
the RNA isolated from the tissue samples described above (for
example using a commercial reverse transcription kit). OPN-c cDNA
is amplified using appropriate primers (for example using primers
having a detectable label), and the resulting OPN-c amplicons
detected. The relative amount of OPN-c amplicons in the tumor
tissue sample can be compared to a reference value, such as a
relative amount of OPN-c amplicons present in the adjacent
non-tumor sample from the subject, wherein the presence of
significantly more OPN-c amplicons in the tumor sample as compared
to the non-tumor sample (such as an increase of at least 2-fold, at
least 3-fold, or at least 5-fold) indicates that the subject has
metastatic HCC, has an increased likelihood of a primary HCC
metastasizing, has a poor prognosis, or combinations thereof.
[0342] In some examples, relative amount of OPN-5 kD protein and
OPN-c mRNA expression are determined in the same subject using the
methods described above.
Example 19
Screening for Small Molecule Inhibitors
[0343] This example provides exemplary methods that can be used to
identify small molecule inhibitors that mimic inhibition observed
with OPN-5 kD specific antibodies.
[0344] A binding assay that measures OPN-5 kD binding to its
antibody (such as 5 dk106-13 D), for example an ELISA, Western
blot, or other immunoassay. Binding of the antibody to OPN-5 kD is
detected in the presence and absence of one or more test compounds.
Such test compounds are commercially available or can be generated
using routine methods. Small molecules that disrupt the binding
between the antibody and the OPN-5 kD fragment are candidate
inhibitors of OPN-5 kD, and thus may be used in the therapeutic
methods provided herein. For example, such molecules may bind to
the paratope of the antibody and hence block its ability to bind to
the OPN-5 kD fragment. Alternatively, the small molecule inhibitors
can also bind to the OPN-5 kD fragment and block the epitope of the
antibody.
[0345] Molecules identified using the methods can be further
analyzed for their inhibition of OPN-5 kD activity, and the ability
to treat an OPN-overexpressing tumor, for example inhibit or reduce
metastasis of a tumor, for example using the methods described in
Examples 12-15.
[0346] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only examples of
the invention and should not be taken as limiting the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
1911424DNAHomo sapiens 1gaccagactc gtctcaggcc agttgcagcc ttctcagcca
aacgccgacc aaggaaaact 60cactaccatg agaattgcag tgatttgctt ttgcctccta
ggcatcacct gtgccatacc 120agttaaacag gctgattctg gaagttctga
ggaaaagcag ctttacaaca aatacccaga 180tgctgtggcc acatggctaa
accctgaccc atctcagaag cagaatctcc tagccccaca 240gaatgctgtg
tcctctgaag aaaccaatga ctttaaacaa gagacccttc caagtaagtc
300caacgaaagc catgaccaca tggatgatat ggatgatgaa gatgatgatg
accatgtgga 360cagccaggac tccattgact cgaacgactc tgatgatgta
gatgacactg atgattctca 420ccagtctgat gagtctcacc attctgatga
atctgatgaa ctggtcactg attttcccac 480ggacctgcca gcaaccgaag
ttttcactcc agttgtcccc acagtagaca catatgatgg 540ccgaggtgat
agtgtggttt atggactgag gtcaaaatct aagaagtttc gcagacctga
600catccagtac cctgatgcta cagacgagga catcacctca cacatggaaa
gcgaggagtt 660gaatggtgca tacaaggcca tccccgttgc ccaggacctg
aacgcgcctt ctgattggga 720cagccgtggg aaggacagtt atgaaacgag
tcagctggat gaccagagtg ctgaaaccca 780cagccacaag cagtccagat
tatataagcg gaaagccaat gatgagagca atgagcattc 840cgatgtgatt
gatagtcagg aactttccaa agtcagccgt gaattccaca gccatgaatt
900tcacagccat gaagatatgc tggttgtaga ccccaaaagt aaggaagaag
ataaacacct 960gaaatttcgt atttctcatg aattagatag tgcatcttct
gaggtcaatt aaaaggagaa 1020aaaatacaat ttctcacttt gcatttagtc
aaaagaaaaa atgctttata gcaaaatgaa 1080agagaacatg aaatgcttct
ttctcagttt attggttgaa tgtgtatcta tttgagtctg 1140gaaataacta
atgtgtttga taattagttt agtttgtggc ttcatggaaa ctccctgtaa
1200actaaaagct tcagggttat gtctatgttc attctataga agaaatgcaa
actatcactg 1260tattttaata tttgttattc tctcatgaat agaaatttat
gtagaagcaa acaaaatact 1320tttacccact taaaaagaga atataacatt
ttatgtcact ataatctttt gttttttaag 1380ttagtgtata ttttgttgtg
attatctttt tgtggtgtga ataa 14242314PRTHomo sapiens 2Met Arg Ile Ala
Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala1 5 10 15Ile Pro Val
Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu 20 25 30Tyr Asn
Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro 35 40 45Ser
Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser Ser Glu 50 55
60Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro Ser Lys Ser Asn Glu65
70 75 80Ser His Asp His Met Asp Asp Met Asp Asp Glu Asp Asp Asp Asp
His 85 90 95Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp
Val Asp 100 105 110Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His
His Ser Asp Glu 115 120 125Ser Asp Glu Leu Val Thr Asp Phe Pro Thr
Asp Leu Pro Ala Thr Glu 130 135 140Val Phe Thr Pro Val Val Pro Thr
Val Asp Thr Tyr Asp Gly Arg Gly145 150 155 160Asp Ser Val Val Tyr
Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg 165 170 175Pro Asp Ile
Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His 180 185 190Met
Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala 195 200
205Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser
210 215 220Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His
Ser His225 230 235 240Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn
Asp Glu Ser Asn Glu 245 250 255His Ser Asp Val Ile Asp Ser Gln Glu
Leu Ser Lys Val Ser Arg Glu 260 265 270Phe His Ser His Glu Phe His
Ser His Glu Asp Met Leu Val Val Asp 275 280 285Pro Lys Ser Lys Glu
Glu Asp Lys His Leu Lys Phe Arg Ile Ser His 290 295 300Glu Leu Asp
Ser Ala Ser Ser Glu Val Asn305 31031343DNAHomo sapiens 3gaccagactc
gtctcaggcc agttgcagcc ttctcagcca aacgccgacc aaggaaaact 60cactaccatg
agaattgcag tgatttgctt ttgcctccta ggcatcacct gtgccatacc
120agttaaacag gctgattctg gaagttctga ggaaaagcag aatgctgtgt
cctctgaaga 180aaccaatgac tttaaacaag agacccttcc aagtaagtcc
aacgaaagcc atgaccacat 240ggatgatatg gatgatgaag atgatgatga
ccatgtggac agccaggact ccattgactc 300gaacgactct gatgatgtag
atgacactga tgattctcac cagtctgatg agtctcacca 360ttctgatgaa
tctgatgaac tggtcactga ttttcccacg gacctgccag caaccgaagt
420tttcactcca gttgtcccca cagtagacac atatgatggc cgaggtgata
gtgtggttta 480tggactgagg tcaaaatcta agaagtttcg cagacctgac
atccagtacc ctgatgctac 540agacgaggac atcacctcac acatggaaag
cgaggagttg aatggtgcat acaaggccat 600ccccgttgcc caggacctga
acgcgccttc tgattgggac agccgtggga aggacagtta 660tgaaacgagt
cagctggatg accagagtgc tgaaacccac agccacaagc agtccagatt
720atataagcgg aaagccaatg atgagagcaa tgagcattcc gatgtgattg
atagtcagga 780actttccaaa gtcagccgtg aattccacag ccatgaattt
cacagccatg aagatatgct 840ggttgtagac cccaaaagta aggaagaaga
taaacacctg aaatttcgta tttctcatga 900attagatagt gcatcttctg
aggtcaatta aaaggagaaa aaatacaatt tctcactttg 960catttagtca
aaagaaaaaa tgctttatag caaaatgaaa gagaacatga aatgcttctt
1020tctcagttta ttggttgaat gtgtatctat ttgagtctgg aaataactaa
tgtgtttgat 1080aattagttta gtttgtggct tcatggaaac tccctgtaaa
ctaaaagctt cagggttatg 1140tctatgttca ttctatagaa gaaatgcaaa
ctatcactgt attttaatat ttgttattct 1200ctcatgaata gaaatttatg
tagaagcaaa caaaatactt ttacccactt aaaaagagaa 1260tataacattt
tatgtcacta taatcttttg ttttttaagt tagtgtatat tttgttgtga
1320ttatcttttt gtggtgtgaa taa 1343444PRTHomo sapiens 4Leu Arg Ser
Lys Ser Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro1 5 10 15Asp Ala
Thr Asp Glu His Ile Thr Ser His Met Glu Ser Glu Glu Leu 20 25 30Asn
Gly Ala Tyr Lys Ala Ile Pro Val Ala Gln Asp 35 40510PRTHomo sapiens
5Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp1 5 10610PRTHomo sapiens
6His Met Glu Ser Glu Glu Leu Asn Gly Ala1 5 10710PRTHomo sapiens
7Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro1 5 10815PRTHomo sapiens
8His Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro1 5 10
159287PRTHomo sapiens 9Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu
Gly Ile Thr Cys Ala1 5 10 15Ile Pro Val Lys Gln Ala Asp Ser Gly Ser
Ser Glu Glu Lys Gln Asn 20 25 30Ala Val Ser Ser Glu Glu Thr Asn Asp
Phe Lys Gln Glu Thr Leu Pro 35 40 45Ser Lys Ser Asn Glu Ser His Asp
His Met Asp Asp Met Asp Asp Glu 50 55 60Asp Asp Asp Asp His Val Asp
Ser Gln Asp Ser Ile Asp Ser Asn Asp65 70 75 80Ser Asp Asp Val Asp
Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser 85 90 95His His Ser Asp
Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp 100 105 110Leu Pro
Ala Thr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp Thr 115 120
125Tyr Asp Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser
130 135 140Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr
Asp Glu145 150 155 160Asp Ile Thr Ser His Met Glu Ser Glu Glu Leu
Asn Gly Ala Tyr Lys 165 170 175Ala Ile Pro Val Ala Gln Asp Leu Asn
Ala Pro Ser Asp Trp Asp Ser 180 185 190Arg Gly Lys Asp Ser Tyr Glu
Thr Ser Gln Leu Asp Asp Gln Ser Ala 195 200 205Glu Thr His Ser His
Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn 210 215 220Asp Glu Ser
Asn Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser225 230 235
240Lys Val Ser Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp
245 250 255Met Leu Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His
Leu Lys 260 265 270Phe Arg Ile Ser His Glu Leu Asp Ser Ala Ser Ser
Glu Val Asn 275 280 2851025DNAArtificial SequenceSynthetic
oligonucleotide primer. 10tctcctagcc ccacagaatg ctgtg
251125DNAArtificial SequenceSynthetic oligonucleotide primer.
11aggaaaagca gaatgctgtg tcctc 251223PRTHomo sapiens 12Asp Ile Val
Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly1 5 10 15Glu Arg
Ala Thr Ile Asn Cys 201314PRTHomo sapiens 13Trp Tyr Gln Gln Lys Pro
Gly Gln Pro Pro Leu Leu Ile Tyr1 5 101432PRTHomo sapiens 14Gly Val
Pro Asp Arg Pro Phe Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu
Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys 20 25
301511PRTHomo sapiens 15Phe Gly Gln Gly Gln Thr Lys Leu Glu Ile
Lys1 5 101630PRTHomo sapiens 16Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gln Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gln Tyr Thr Phe Thr 20 25 301714PRTHomo sapiens 17Trp Val Arg
Gln Ala Pro Gly Gln Arg Leu Glu Trp Met Gly1 5 101832PRTHomo
sapiens 18Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr
Met Glu1 5 10 15Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys Ala Arg 20 25 301911PRTHomo sapiens 19Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser1 5 10
* * * * *